Biosynthesis of the Aromatic Amino Acids
Chapter
28
A. J. PITTARD
The pathway of biosynthesis of the aromatic amino acids is shown, for convenience, in four parts (Fig. 1 through 4). Figure 1 shows the "common pathway" leading to the synthesis of the branch point compound chorismate, and Figures 2, 3, and 4 show the three terminal pathways in which chorismate is converted to phenylalanine, tyrosine, and tryptophan, respectively. The common pathway has sometimes been referred to as the shikimate pathway. An additional four terminal pathways lead from chorismate to folate, ubiquinone, menaquinone, and enterochelin, but these are not considered here.
Identification of the various intermediates in the aromatic pathway was completed by the early 1960s. As with other pathways, studies of auxotrophic mutants were of key importance. The first intermediate to be identified was shikimate; its position in the common pathway was established when Davis (95) showed that this compound alone of 55 aromatic and hydroaromatic compounds tested could fulfill the requirement of certain aromatic auxotrophs of Escherichia coli for tyrosine, phenylala- nine, and tryptophan. Other mutants, which failed to respond to shikimate, accumulated this compound in their culture fluids. In the same study, it was established that anthranilate could be substituted for tryptophan, phenylpyruvate could be substituted for phenylalanine, and under appropriate conditions, 4-hydroxyphenyl pyruvate could be substituted for tyrosine. The accumulation by auxotrophs of intermediates preceding the blocked reaction led to the identification of 3-dehydroquinate (DHQ), 3-dehydroshikimate, and shikimate 3-phosphate, all intermediates in the common pathway (100, 279, 326, 328).
The identification of a phosphorylated compound (shikimate 3-phosphate) in the culture fluids was an exception to the general experience with other phosphorylated intermediates in which the compounds that accumulated in culture fluids were predominantly in the dephosphorylated form. Such was the case with enol-pyruvyl shikimate (EPS), indoleglycerol (InG), and 1-(o-carboxyphenylamino)-1-deoxyribulose (99, 111, 138, 271). Neither the phosphorylated intermediates nor their dephosphorylated derivatives serve as growth factors for any mutant blocked prior to their formation in a pathway, since the phosphorylated compounds cannot enter the cell, and the dephosphorylated derivatives are generally not true intermediates. Shikimate is a notable exception. The branch point compound chorismate fails to act as a growth factor, presumably because it is not able to enter the cell.
The early reactions in the common pathway were more difficult to establish. A mutant strain of E. coli that accumulated shikimic acid was grown on glucose variously labeled with 14C. The shikimate was isolated and chemically degraded to establish the distribution of specific carbon atoms of glucose in the shikimate molecule. The results showed that three of the carbon atoms of shikimate were derived from glycolysis and four were derived from the pentose phosphate pathway (308).
The nature of these precursor compounds was established when erythrose 4-phosphate became available (22) and it was demonstrated that cell extracts of E. coli could convert erythrose 4-phosphate and phosphoenolpyruvate (PEP) to DHQ. Furthermore, on fractionation, an enzyme preparation could be obtained that converted erythrose 4-phosphate and PEP to 2-keto-3-deoxy-d-arabo-heptonic acid 7-phosphate (306). This compound was later to be termed 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP).
The identification of chorismate, the branch point compound, required the use of mutant strains blocked in each of the three terminal pathways. A strain of Aerobacter aerogenes (62–1) that was blocked in the second reaction of the tryptophan pathway and the first reactions of both the phenylalanine and the tyrosine pathways was isolated. Extracts of this strain were able to convert shikimate to anthranilate. When, however, glutamine was omitted from the reaction mixture, a new compound was formed. This compound could be extracted from the reaction mixture and used as a substrate for anthranilate production by extracts of a mutant blocked after the EPS phosphate (EPSP) step (140). Subsequent studies showed that the compound could be accumulated by strain 62–1 and that it could be converted enzymically into anthranilate, prephenate, 4-hydroxyphenylpyruvate, phenylpyruvate, and 4-hydroxybenzoate. The compound was called chorismic acid (137, 140). After the structure of chorismate had been established, it was shown that it could be formed from EPSP by cell extracts of E. coli (239). The position of EPSP in the pathway was further supported by the demonstration that cell extracts produced EPSP from shikimate 3-phosphate and PEP and could convert EPSP to phenylpyruvate (213).
All of the reactions shown in Fig. 1 through 4 have been confirmed in cell extracts; many of these experiments used purified enzyme preparations. Although much of the early work establishing the pathway used E. coli and in some cases A. aerogenes, the reactions apply equally to Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium).
Srinivasan and Sprinson (309) were the first to demonstrate the conversion of erythrose 4-phosphate and PEP to DAHP in cell extracts of E. coli. It was subsequently shown (300) that this activity comprised at least two enzymes that were distinguishable by their inhibition and repression by phenylalanine and tyrosine, respectively. A few years later, a minor third enzyme was identified whose synthesis was repressed by tryptophan (42, 110). This enzyme was subsequently shown to be subject to inhibition by tryptophan (57, 109, 258, 269). Whereas inhibition of DAHP synthase (TYR) and DAHP synthase (PHE) by tyrosine and phenylalanine was as high as 95%, the inhibition of DAHP synthase (TRP) by tryptophan did not exceed 60% (259). Early studies in E. coli of the reaction mechanisms of DAHP synthase (PHE) and DAHP synthase (TYR) involved enzyme preparations with low specific activities. Results with those preparations, which suggested a ping-pong mechanism of reaction and gave Kms in the molar range (237, 312), were later disproved when more highly purified enzyme preparations were used. With a highly purified preparation of DAHP synthase (TYR) from S. typhimurium, the reaction mechanism was ordered and sequential rather than ping-pong, with PEP being the first substrate to bind (106). Similar results were obtained with a highly purified preparation of DAHP synthase (PHE) from E. coli K-12 (299) and with DAHP synthase (TYR) purified to homogeneity from E. coli K-12 (291). A Km of 5.8 μM for PEP was reported for purified DAHP synthase (TYR) (291), and although data were not presented, a similar value was suggested for purified DAHP synthase (PHE) (228). In conflict with these results is a value of 80 ± 40 μM obtained with highly purified DAHP synthase (PHE) (298). A value of 14.9 μM has been reported for the partially purified DAHP synthase (TYR) of S. typhimurium (106). It has been pointed out (228) that because the intracellular concentration of PEP in E. coli never falls below 88 μM (218), the enzyme-PEP complex may be the native form of both DAHP synthase (TYR) and DAHP synthase (PHE).
The three enzymes from E. coli K-12 and DAHP synthase (TYR) from S. typhimurium have been purified to homogeneity. DAHP synthase (TYR) and DAHP synthase (TRP) are dimers with subunit molecular weights of about 40,000 (173, 268, 291; M. D. Poling, J. Suzick, J. Shultz, and K. M. Herrmann, Fed. Proc. 40:1581, 1981). DAHP synthase (PHE), on the other hand, is a tetramer with a subunit molecular weight of 35,000 (228).
The genes for these three enzymes were identified by mutation and mapped (107, 320, 321). Subsequently, they were cloned in E. coli, and the complete nucleotide sequences were determined (94, 174, 176, 269, 295, 360). The complete amino acid sequence of DAHP synthase (TYR) was also determined (295). The sequence suggests that the three isoenzymes have a common evolutionary origin (269). Earlier studies which had compared only the first 40 amino acids at the amino termini of DAHP synthase (TRP), DAHP synthase (TYR), and DAHP synthase (PHE) had led to the opposite conclusion that the three enzymes had evolved independently and not from a common precursor (Poling et al., Fed. Proc., 1981). Comparison of the entire sequence (295), however, coupled with the finding that sites affecting catalytic activity or feedback sensitivity were distributed throughout DAHP synthase (TRP) instead of being located in specific domains reinforced the theory of a common evolution (269). All three DAHP synthases have been shown to be metalloenzymes, and DAHP synthase (PHE) has been shown to contain 1 mol of iron per mol of tetramer (227).
There are conflicting reports as to whether the metal in DAHP synthase (TYR) is Cu2+ or Fe2+ (15, 227), whereas DAHP synthase (TRP) appears to be activated only by Fe2+ (268). Although DAHP synthase (TYR) contains a short amino acid sequence which is highly homologous to a putative iron-binding sequence in hemerythrin, the oxygen-carrying molecule in the sea worm Phascolopsis gouldii (167), this sequence is not conserved in DAHP synthase (TRP) and DAHP synthase (PHE) (296).
The presence of three isofunctional DAHP synthase enzymes with activities and rates of synthesis differentially affected by individual amino acids provides the cell with an ability to modulate the overall rate of synthesis in response to changes in the availability of particular aromatic amino acids. In addition to the feedback inhibition of the three enzymes by the respective amino acids, the synthesis of DAHP synthase (TYR) is repressed by tyrosine or very high levels of phenylalanine, that of DAHP synthase (PHE) is repressed by phenylalanine and tryptophan, and that of DAHP synthase (TRP) is repressed by tryptophan (44). Furthermore, mutations in the gene for tryptophanyl- tRNA synthase (trpS) have been shown to affect aroF expression, even in derepressed strains (53).
Mutations conferring feedback resistance on DAHP synthase (TRP) (269) and on DAHP synthase (TYR) (324) have been characterized: two amino acid substitutions in DAHP synthase (TRP) (at positions 147 and 149) and one in DAHP synthase (TYR) (at position 148) confer feedback resistance. It has been postulated (324) that this region in the enzyme makes up part of an amino acid-binding pocket.
When E. coli grows in minimal medium, DAHP synthase (PHE) makes up about 80% or more of the total DAHP synthase activity; however, under conditions in which the aromatic amino acids limit growth, as in minimal medium supplemented with all amino acids except phenylalanine, tyrosine, and tryptophan, derepression of DAHP synthase (TYR) makes it the major enzyme (314). DAHP synthase (PHE) is also the major isoenzyme present when S. typhimurium is grown in minimal medium (106, 173), although there is one report of high levels of DAHP synthase (TYR) under these conditions (188). In both organisms, DAHP synthase (TRP) normally accounts for a very small proportion of DAHP synthase activity.
When fully derepressed, the specific activity of DAHP synthase (TRP) in cell extracts is only 50 mU/mg of protein, whereas the corresponding values for DAHP synthase (TYR) and DAHP synthase (PHE) are 560 and 300 mU/mg of protein (in international units, 1 U is 1 μmol of substrate used or product formed per min at 37°C) (56, 314). This difference may be due to a relatively inefficient promoter for aroH, the gene for DAHP synthase (TRP) (360).
Hudson et al. (176) identified two active promotors for aroH. Expression from both of them is repressed by tryptophan, but expression from the weaker of the two, P2, is stimulated about 13-fold in a yeast extract tryptone medium. The stimulated expression from P2, however, is still less than the expression from P1 in minimal medium.
In vivo studies with mutant strains containing single isoenzymes have shown that in the presence of all three aromatic amino acids, the cell retains enough residual activity of either DAHP synthase (PHE) or DAHP synthase (TRP) to allow for continued synthesis of the aromatic vitamins (323). When cells reach stationary phase, DAHP synthase (TYR) activity decays much faster than DAHP synthase (PHE) activity, presumably as a result of some specific degradation process (315; A. DeLucia, R. Schoner, and K. M. Herrmann, Int. Congr. Biochem., abstr. 11, p. 252, 1979).
DHQ synthase, formerly known as 5-dehydroquinate synthetase, catalyzes the conversion of DAHP to DHQ. DHQ was first identified as an intermediate in aromatic biosynthesis in 1953 (326). It was isolated from the culture fluids of an aromatic auxotroph of E. coli. The active compound had previously been shown to support the growth of mutants blocked very early in the aromatic pathway. Its structure was determined, and it was shown to be chemically convertible to dehydroshikimate by heat at an acid pH (100).
The enzymic conversion of DAHP to DHQ was first reported in 1963 with a partially purified enzyzme preparation from E. coli. The enzyme required both NAD+ and Co2+ for activity, and a scheme was proposed to account for the molecular conversion of DAHP to DHQ. Key steps in this scheme were the oxidation and later reduction of the C-5 of DAHP by processes involving NAD (307).
In 1964, 3,7-dideoxy-d-threo-hepto-2,6-diulosonic acid, the compound proposed to be the immediate precursor of DHQ, was synthesized. Although this compound could be converted chemically at pH 11 to DHQ, it was not converted to DHQ enzymically (3). Subsequent studies by Rotenberg and Sprinson (276, 277) and LeMarechal and Azerad (210) with DAHP specifically labeled with tritium have established that this diketo compound is not formed as an intermediate in the formation of DHQ. A methyl group is never formed at C-7, and the cyclization step involves an interaction between the enol formed in phosphate elimination and the carbonyl at C-2 in an aldolase-type reaction. Kinetic isotope effects were observed at C-5, supporting a mechanism involving oxidation at C-5 by NAD. The finding that all the tritium of labeled DAHP is conserved in DHQ establishes that hydride transfer in the subsequent reduction of C-5 involves the same hydrogen atom that was taken from DAHP. NADH is enzyme bound and reduces the keto group at C-5 with the regeneration of NAD (211, 276, 277).
DHQ synthase has been purified to homogeneity from E. coli B and shown to be a single polypeptide with a molecular weight of 57,000. The native enzyme is a monomer, and the Km for DAHP is 33 μM (220). Berlyn and Giles (33) measured the molecular weight of the DHQ synthase enzyme of E. coli K-12 by using ultracentrifugation in sucrose density gradients and found it to be 56,000. Frost et al. (126) used gene-cloning techniques to produce high levels of DHQ synthase for purification. They purified the enzyme to homogeneity and showed it to be a monomeric protein with an M r between 40,000 and 44,000.
Recently, the structural gene for this enzyme (aroB) was cloned, and its sequence was determined; the protein was overexpressed, and its amino acid composition and amino-terminal sequence were determined. Judging from the sequence, the monomer is a protein of 362 amino acids with a calculated M r of 38,800 (234).
As with the other remaining enzymes of the common pathway (with the exception of shikimate kinase), DHQ synthase appears to be synthesized constitutively (314). Its synthesis is not repressed by any of the aromatic amino acids or by chorismate, nor is it induced by DAHP. The specific activity of DHQ synthase in extracts of E. coli is 25 mU/mg of protein. It has been calculated that this is about fivefold greater than would be required to meet the aromatic needs of cells growing with a doubling time of about 1 h (314). Ogino et al. (250) demonstrated that strains of E. coli with feedback-resistant DAHP synthase (TYR) accumulate DAHP, indicating that under these conditions, DHQ synthase activity has become rate limiting.
DHQ dehydratase, formerly known as dehydroquinase, catalyzes the conversion of DHQ to 3-dehydroshikimate and introduces the first double bond of the aromatic ring. The reaction exhibits cis stereochemistry in both directions (46, 156, 319). The enzymic reaction was first studied by Mitsuhashi and Davis, who partially purified the enzyme from both A. aerogenes and E. coli. They separated DHQ dehydratase from dehydroshikimate reductase (now known as shikimate dehydrogenase) and demonstrated the reversibility of the reaction. The Km for DHQ was 44 μM (236). Some confusion existed concerning the molecular weight of the enzyme. Berlyn and Giles (33) reported a molecular weight of 40,000 for the native enzyme from E. coli. However, Kinghorn et al. (195), using the same technique of ultracentrifugation in sucrose density gradients, reported a molecular weight of about 59,000 for native enzyme from wild-type E. coli K-12 and from a derivative that carried the gene for DHQ dehydratase on plasmid pBR322. The use of minicells to study plasmid-encoded proteins revealed a protein band with a molecular weight of 63,000 which appeared to represent native DHQ dehydratase. A 31,000-molecular-weight band that appeared coincidentally was hypothesized to represent a subunit of the native enzyme (195). More recently, Chaudhuri et al. (61) reported that DHQ dehydratase from E. coli is a simple dimeric protein with a subunit M r of 29,000. The structural gene (aroD) was cloned, and its sequence was determined. The protein was overexpressed, and its amino acid composition and amino-terminal sequence were determined. This information plus the DNA sequence indicated that aroD encodes a polypeptide of 240 amino acids with a calculated M r of 26,377 (115). As previously mentioned for DHQ synthase, DHQ dehydratase appears to be synthesized constitutively. When E. coli and S. typhimurium are grown in minimal medium, DHQ dehydratase specific activities range from 50 to 100 mU/mg (146, 195, 261, 314).
In their original paper, Mitsuhashi and Davis (236) made the observation that mutants of A. aerogenes selected for good growth on quinate and a mutant of E. coli blocked between 3-dehydroshikimate and shikimate produced greatly enhanced levels of DHQ dehydratase. This latter observation was not confirmed in the study of DHQ dehydratase levels in aromatic auxotrophs of E. coli and S. typhimurium (146, 261).
The enzyme that converts 3-dehydroshikimate to shikimate was first studied in 1954 with partially purified extracts of E. coli W. The reaction was studied in the reverse direction (shikimate to dehydroshikimate) in a coupled reaction with glutathione reductase to oxidize the NADPH that is formed. The reaction is dependent on NADP and specific for shikimate. Shikimate dehydrogenase activity was not detected in extracts of aromatic auxotrophs which accumulated dehydroskimate. The Km of the enzyme for shikimate was 55 μM (342). The reaction is stereospecific, involving transfer of hydrogen from the A side of NADPH (92). Estimation of molecular weight by sucrose gradient centrifugation gives a value of 25,000 (33). Recently, Chaudhuri and Coggins (60) purified shikimate dehydrogenase from E. coli to homogeneity and reported that it is a monomeric protein with an M r of 32,000. The structural gene (aroE) was cloned, and its sequence was determined. The amino acid compositon and amino-terminal sequence of the purified protein were determined. These results indicate that the monomer is a polypeptide of 272 amino acids with a calculated M r of 29,380 (11). The constitutive level of this enzyme in cells grown in minimal medium is about 60 mU/mg (314).
Shikimate kinase catalyzes the formation of shikimate 3-phosphate from shikimate and ATP. Shikimate 3-phosphate was first identified as a possible intermediate in aromatic biosynthesis by Davis and Mingioli (99). Its chemical structure was established by Weiss and Mingioli (328). No mutants blocked in the shikimate kinase reaction were found among aromatic auxotrophs of either E. coli (261) or S. typhimurium (146). The reasons for this became apparent when it was shown that extracts of S. typhimurium yield two separable peaks of shikimate kinase activity when they are chromatographed on DEAE-cellulose (240). Using ultracentrifugation in sucrose density gradients, Berlyn and Giles (33) confirmed the observation made in Salmonella spp. and established that E. coli also appears to have two shikimate kinase enzymes. Further study of the E. coli enzymes established that the synthesis of one of these (called shikimate kinase II because it was the second shikimate kinase to be eluted from DEAE-Sephadex) was subject to specific control. In particular, when cells were starved for tyrosine and tryptophan or when they carried inactive tyrR regulator genes, synthesis of shikimate kinase II was derepressed about 10-fold. Shikimate kinase levels vary between 5 and 55 mU/mg. A mutant lacking shikimate kinase II activity was isolated (122). The structural gene for shikimate kinase II (aroL) was cloned, and its sequence was determined (103, 104, 235). The protein translated from the sequence has an M r of 18,998. Recently, the gene for shikimate kinase I (aroK) was identified as part of a transcription unit including aroB. The aroK sequence was reported to show a 34% homology to shikimate kinase II in a 97-amino-acid region (217). A reexamination of this sequence identified some errors, and the revised amino acid sequence for shikimate kinase I shows a 30% homology with shikimate kinase II over the entire length of both proteins (173 and 174 amino acids, respectively [330a]). Shikimate kinase II was purified to homogeneity, and the amino acid sequence of its amino terminus was determined (97, 235). Shikimate kinase I was partially purified (358-fold from cell extracts), and some of its physical parameters were measured (R. C. De Feyter, Ph.D. thesis, University of Melbourne, Melbourne, Australia, 1984).
An examination of the kinetics of the enzymic reaction with fully purified shikimate kinase II and partially purified shikimate kinase I revealed important differences between the two enzymes. The Km of shikimate kinase II for shikimate is 200 μM. On the other hand, the Km of shikimate kinase I for shikimate appears to be in excess of 5 mM. This apparent low affinity of shikimate kinase I for shikimate explains why aroL mutants which lack shikimate kinase II activity will grow in minimal media only if the levels of DAHP synthase are high. Neither enzyme is inhibited by any of the end products, and shikimate kinase II requires Mg2+ as a cofactor (105).
The existence of isofunctional enzymes has been observed most frequently in the first reaction of a pathway that later branches to multiple end products, e.g., aspartokinases for lysine, methionine, and threonine (70) and DAHP synthases for the aromatic amino acids. It is also true that specific regulation of gene expression most frequently affects genes specifying the first enzyme in a pathway, and it is noteworthy that of all the genes of the common pathway of aromatic biosynthesis, only the three specifying the three DAHP synthase enzymes and the gene for shikimate kinase II are subject to control.
Does this mean that in the evolutionary past, aromatic biosynthesis started with shikimate, or does it indicate an as yet undiscovered pathway diverging from shikimate? Although Gollub et al. (146) observed no regulation of shikimate kinase activity in S. typhimurium, cells had not been grown under conditions of starvation for tyrosine and tryptophan, and the question as to whether S. typhimurium and E. coli differ remains to be established.
The very low affinity of shikimate kinase I for shikimate raises the possibility that this enzyme has another major function, although mutational inactivation of aroK produces a detectable phenotype only in an aroL mutant background (217).
EPSP synthase converts PEP and shikimate 3-phosphate into EPSP. It was the dephosphorylated form of this compound that was first identified in culture fluids of aromatic auxotrophs blocked in the last step of the common pathway (99).
Levin and Sprinson (212, 213) partially purified the enzyme activity from E. coli and showed that the product of the reaction was EPSP. The mechanism of this reaction was studied with partially purified enzyme from Salmonella spp. and shown to involve the transfer of the enolpyruvyl grouping unchanged to the acceptor molecule (37), as was first proposed in a scheme by Levin and Sprinson (213). This reaction introduces the three-carbon fragment that is destined to become the side chain of phenylalanine and tyrosine but to be replaced in the synthesis of tryptophan. The enzyme from E. coli has been purified to homogeneity (214). The subunit M r was estimated to be 49,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the native molecular weight was estimated to be 55,000 by gel filtration, confirming that the enzyme is a monomer. A key step in the purification was elution with substrate from a cellulose phosphate column. A mixture of shikimate 3-phosphate and PEP was very effective, whereas PEP alone had no effect, and shikimate 3-phosphate alone caused elution over a very broad peak. The researchers who accomplished this purification concluded that these results agree with an ordered reaction in which shikimate 3-phosphate binds to the enzyme first.
The gene for this enzyme, aroA, has been cloned, and strains carrying this gene on a multicopy plasmid overproduce EPSP synthase 100-fold (117). The Kms for PEP and shikimate 3-phosphate are 16 and 2.5 μM, respectively. The enzyme isolated from the overproducing strain (116) is indistinguishable from that produced by wild-type E. coli. The amino-terminal sequence and the amino acid composition of the purified protein have been determined. This information, in conjunction with the nucleotide sequence of the aroA gene, indicates that the primary translation product is a polypeptide of 427 amino acids with a calculated M r of 46,112 (116).
A molecular analysis of the region containing aroA revealed that this region is in a transcription unit with serC. serC is the first gene in the transcription unit, and some transcripts terminate between serC and aroA (114; K. Duncan, A. Lewendon, and J. R. Coggins, Biochem. Soc. Trans. 14:263–264, 1986). Genetic analyses of various S. typhimurium mutants lacking EPSP synthase has revealed that in that organism also, serC and aroA constitute a unit of transcription (171). The aroA gene from S. typhimurium was cloned, and its sequence was determined (311).
The enzyme EPSP synthase is synthesized constitutively in S. typhimurium (146) and E. coli (314), and cell extracts have a specific activity of about 100 mU/mg of protein. Since the phosphoserine aminotransferase specified by serC is also synthesized constitutively (263), it is not clear that the cell gains any particular advantage from having serC and aroA in a single transcription unit.
The chorismate synthase reaction introduces a second double bond into the aromatic ring system and forms the branch point compound chorismate, which serves as the starting substrate for the three terminal pathways of aromatic amino acid biosynthesis and for the pathways to ubiquinone, menaquinone, folate, and enterochelin (137, 140). The enzyme has been partially purified from E. coli. It is oxygen sensitive and requires a reducing environment that can be established with an FADH2- and NADH-regenerating system in an anaerobic environment. Activity is inhibited by iron chelators and by high levels of Fe2+. The basis for the latter effect is unknown, but it is postulated that Fe2+ is a cofactor for chorismate synthase. It is suggested that the reaction is irreversible. A tentative molecular weight for the enzyme of somewhere between 70,000 and 100,000 has been proposed (239).
There are two reports of the cloning and sequence analysis of the gene for chorismate synthase of E. coli (59, 331). A difference between the two nucleotide sequences results in different predicted amino acid sequences for the 36 amino acids at the carboxyl end of the protein. Removal of these amino acids does not destroy enzyme activity, so the reported difference may be a true polymorphism The calculated M r for the monomer from E. coli is either 39,138 or 38,183 (59, 331), and the M r for the monomer from S. typhimurium is 39,108 (59). Gel filtration experiments show that the native enzyme is a tetramer (331).
Synthesis of the enzyme is believed to be constitutive in both S. typhimurium and E. coli. The specific activity of chorismate synthase in cell extracts is only 10 to 20% of that of the prior enzyme, EPSP synthase (146, 314).
It is puzzling that the last reaction of the pathway appears to be rate limiting, unless such a situation facilitates subsequent controls directing chorismate along the various terminal pathways. Clearly, more work needs to be done on this important enzyme and its role in vivo in aromatic biosynthesis.
The first reaction of both the phenylalanine and the tyrosine pathways involves the conversion of chorismate to prephenate. The structure of prephenate was determined by Weiss et al. (327). This relatively unstable compound can be converted to phenylpyruvate at an acid pH. This reaction provided some puzzling results when phenylalanine auxotrophs were first studied.
Simmonds (298) described a phenylalanine auxotroph that appeared on prolonged culture to provide its own auxotrophic requirements. Davis (98) and Katagiri and Sato (193) investigated this phenomenon and suggested that the substrate of the blocked reaction was being converted chemically to a precursor in phenylalanine biosynthesis. Davis showed that the phenylalanine precursor is phenylpyruvate and the accumulated substrate is prephenate. The enzymes that carry out the synthesis of prephenate have been referred to as chorismate mutases. Cotton and Gibson (77) showed that chromatography of cell extracts of A. aerogenes and E. coli on DEAE-cellulose gave two well-separated proteins with chorismate mutase activity. Prephenate dehydratase activity was associated with the first component to be eluted, and prephenate dehydrogenase activity was associated with the second. Prephenate dehydratase carries out the second reaction of the phenylalanine pathway, and prephenate dehydrogenase carries out the second reaction in the tyrosine pathway. Subsequent purifications of these enzymes have confirmed that in each case, both activities are the product of a single bifunctional enzyme (108, 134, 177, 199, 200, 280, 290).
The phenylalanine enzyme is referred to as chorismate mutase-prephenate dehydratase, and the tyrosine enzyme is referred to as chorismate mutase-prephenate dehydrogenase. Because of many similarities between these enzymes, they are discussed together. Both enzymes are homodimers with subunit molecular weights of about 40,000 (21, 93, 134, 177, 199, 200, 280). A number of studies indicate that chorismate mutase-prephenate dehydratase has two independent catalytic sites. For both E. coli and S. typhimurium, mutants in which only one of the catalytic activities has been lost have been isolated (19, 101, 261, 288). The two enzymic activities can be inhibited differentially by chemical modifying reagents (135, 136, 178), by phenylalanine (108), and by substrate analogs (20). Kinetic studies and the use of radiolabeled chorismate suggest that when the reaction is carried out with purified enzyme in vitro, prephenate dissociates from the mutase site and equilibrates with the bulk medium before combining at the dehydratase site (113). Kinetic studies also support a random mechanism involving the formation of two dead-end complexes, E-NADH-prephenate and E-NAD-hydroxyphenyl pyruvate (281).
Chorismate mutase-prephenate dehydrogenase mutants that have lost dehydrogenase activity but retained mutase activity have been isolated (101, 273). Although chemical modification of the purified enzyme causes parallel loss of both activities (177, 201), tyrosine differentially inhibits prephenate dehydrogenase activity (175), NAD activates chorismate mutase activity, and chorismate stimulates prephenate dehydrogenase activity (169). Kinetic and computer simulation studies, on the other hand, support a single active site (168). In the light of all these results, it seems likely that the two reactions of chorismate mutase-prephenate dehydrogenase are catalyzed at closely situated and interacting active sites (177).
Chorismate mutase-prephenate dehydrogenase and chorismate mutase-prephenate dehydratase activities are inhibited by tyrosine and phenylalanine, respectively, with the most significant inhibition being exerted on the second activity in both cases. Tyrosine can cause up to 95% inhibition of prephenate dehydrogenase activity and, in the presence of NAD, up to 45% inhibition of the associated chorismate mutase activity (175). Inhibition of prephenate dehydratase by phenylalanine approaches 90% in both E. coli and S. typhimurium, and the associated mutase activity in both cases is inhibited 55% (108, 289, 290). The synthesis of chorismate mutase-prephenate dehydrogenase is repressed when cells are grown in the presence of tyrosine. Derepressed synthesis of chorismate mutase-prephenate dehydratase occurs when cells are starved for phenylalanine. This derepression is discussed later.
Because each of these enzymes and anthranilate synthase compete with each other for the same substrate, chorismate, it is of interest to compare the affinities of each for this substrate. The Kms of highly purified enzyme preparations are as follows. Chorismate mutase-prephenate dehydratase from E. coli has a Km for chorismate of 45 μM (108), whereas chorismate mutase-prephenate dehydrogenase from the same organism has Kms for chorismate of 92 μM and for prephenate of 50 μM (177). In contrast, the Km of anthranilate synthase for chorismate is 1.2 μM (17). This very marked difference in affinities implies that under conditions of chorismate limitation, this compound would be preferentially directed down the tryptophan pathway rather than toward phenylalanine or tyrosine. This difference may also explain why aromatic auxotrophs with incomplete blocks in any of the common pathway reactions seem able to meet their requirement for tryptophan but not their requirement for phenylalanine and tyrosine (96).
The last reaction in both the phenylalanine and the tyrosine pathways involves transamination of the respective α-ketoacids, phenylpyruvate, and 4-hydroxyphenylpyruvate, with glutamate as the amino donor. Early studies on aminotransferases in E. coli indicated that there are a number of aminotransferase enzymes and that these enzymes have rather broad specificities (62, 73, 224, 225, 238, 278, 297). Phenylalanine and tyrosine could be formed by at least two enzymes, of which one was termed the aromatic aminotransferase and the other was termed the aspartate aminotransferase. Synthesis of the aromatic aminotransferase could be repressed by tyrosine, but synthesis of the aspartate aminotransferase appeared to be constitutive. There was at least one more enzyme that could function in the conversion of phenylpyruvate to phenylalanine. The analysis of this rather complex situation has been greatly assisted by the isolation of mutants lacking individual aminotransferase enzymes (132, 133, 265, 318) coupled with the development of methods for the separation of aminotransferase activities (224, 225, 266).
Three enzymes need to be considered: the branched-chain amino acid aminotransferase coded for by ilvE, the aromatic aminotransferase whose official name is tyrosine aminotransferase, coded for by tyrB, and the aspartate aminotransferase coded for by aspC. tyrB and aspC double mutants require tyrosine but not phenylalanine for growth, whereas mutants lacking all three activities require both phenylalanine and tyrosine (133). The purification of the aspartate and the aromatic aminotransferase enzymes provides an opportunity to compare their properties. This has been done by Mavrides and Orr (225) and by Powell and Morrison (266). Both studies found that the enzymes are homodimers with subunit molecular weights of approximately 46,000 for the aromatic aminotransferase and 43,000 for the aspartate aminotransferase. Although there are differences in the two reports, both conclude that the amino acid compositions of the two enzymes are very similar.
The aspartate aminotransferase has a much higher affinity for aspartate than does the aromatic aminotransferase. On the other hand, the affinity of the aromatic aminotransferase for phenylalanine and tyrosine and their α-ketoacids is much greater than that of the aspartate aminotransferase. These values, taken from the paper of Powell and Morrison (266), are presented in Table 1. The aminotransferase reaction is freely reversible, and it seems that under normal physiological conditions, phenylalanine and tyrosine syntheses are primarily carried out by the aromatic aminotransferase, the product of the tyrB gene. Only when the pools of phenylpyruvate and 4-hydroxyphenylpyruvate become very large will the aspartate aminotransferase contribute to the synthesis of these two amino acids. The converse is true when one considers the synthesis of aspartate. It is not known whether the ilvE-encoded aminotransferase is ever involved in phenylalanine biosynthesis apart from that seen in tyrB aspC mutant strains. Mavrides and Orr (225) treated purified aromatic aminotransferase with subtilisin and obtained a smaller protein with enhanced aspartate aminotransferase activity. The implication that aspartate aminotransferase is formed by the processing of the aromatic aminotransferase is, however, not supported on a number of grounds (266). Recently, the nucleotide sequences of aspC, ilvE, and tyrB have been determined (125, 205, 206). These studies resolved previous speculations about possible relationships between aspC and tyrB and their encoded aminotransferases. The coding regions of aspC and tyrB encode 396 and 397 amino acid residues, respectively. These sequences have 169 residues in common, and it seems reasonable to assume that gene duplication played a role in their evolution. The deduced amino acid sequence for the aspartate aminotransferase also agrees with the published amino acid sequence for this enzyme purified from E. coli B (203). The gene for the branched-chain aminotransferase ilvE encodes a smaller protein of 309 amino acids and does not show homology to either aspC or tyrB (205). A comparison of the amino acid sequences of the cytoplasmic and mitochondrial aspartate aminotransferases from pig heart with the aspartate aminotransferase from E. coli shows a remarkable 120 invariant residues. Ninety-two of these residues are also present in the bacterial aromatic aminotransferase. Kondo et al. (203) argue that the homologies between the bacterial aspartate aminotransferase and each of the pig heart enzymes marginally favor a hypothesis in which the genes for the bacterial enzyme are more closely related to the gene for the mitochondrial enzyme than to the gene for the cytoplasmic enzyme. The surprising feature overall is that it appears that the postulated duplication of an ancestral bacterial gene to form the precursors of aspC and tyrB occurred very early in evolution, not long after the postulated origin of eukaryotic cells. The relationship between the AspC and TyrB proteins and the bacterial and mammalian enzymes is discussed in a paper by Fotheringham et al. (125), which also contains a detailed discussion of the possible roles of some of the conserved amino acids.
Table 1Kms and Vmaxs for aromatic and aspartate aminotransferases |
Using highly purified preparations of the aromatic aminotransferase, chorismate mutase-prephenate dehydrogenase, and chorismate mutase-prephenate dehydratase, Powell and Morrison (267) demonstrated protein interactions between the aminotransferase and each of the other two enzymes. This interaction did not occur if the aspartate aminotransferase was substituted for the aromatic aminotransferase. The complex with mutase-dehydrogenase dissociated in the presence of tyrosine, and the interaction with mutase-dehydratase required the presence of phenylpyruvate. The interactions therefore are quite specific. The extent to which these interactions occur in vivo and the role they play in synthesis are yet to be determined.
As shown in Fig. 4, the first reaction of the terminal pathway of tryptophan biosynthesis involves the conversion of chorismate and glutamine to anthranilate, glutamate, and pyruvate. The enzyme that carries out this reaction is anthranilate synthase (17, 140). Much of the early work has been reviewed elsewhere (356). The active enzyme is an aggregate composed of two molecules of each of the polypeptides specified by the trpE and trpD genes. These are referred to as component I and component II, respectively. The necessity for both components in the reaction was established in both E. coli and S. typhimurium by studying mutants with nonsense mutations in these genes (30, 121, 186). The aggregate has been purified from both E. coli and S. typhimurium (164, 165, 178, 186, 313). Component I has been purified from E. coli and S. typhimurium, and component II has been purified from S. typhimurium (164, 165, 184, 245, 349). Component I has a molecular weight of about 60,000 and contains the binding site for chorismate. In the absence of component II, component I cannot catalyze the formation of anthranilate using glutamine as the nitrogen source. It can, however, form anthranilate with a considerably reduced efficiency if provided with ammonia instead of glutamine. Component II also has a molecular weight of about 60,000 and specifies two activities. The first of these, a glutamidotransferase activity, is required to activate component I in the anthranilate synthase reaction. This activity channels the nitrogen from glutamine to the active site for anthranilate production. Only the aggregate exhibits this activity, which is stimulated by chorismate. Glutamine hydrolysis requires the prior binding of chorismate to the aggregate (253).
The second activity of component II converts anthranilate to anthranilate-5-phosphoribosyl pyrophosphate. It is termed anthranilate phosphoribosyl (PRA) transferase. This activity can be carried out either by the aggregate or by purified component II.
When anthranilate synthase from S. typhimurium is subjected to limited proteolysis with trypsin, PRA transferase activity is lost, and the aggregate is reduced in size from about 261,000 to about 141,000. Analysis of this lower-molecular-weight aggregate shows that component II has been reduced to a polypeptide with a molecular weight of about 15,000 to 19,000. The aggregate with this reduced component II nevertheless retains anthranilate synthase activity, including glutamidotransferase activity (178). Studies of various trpD nonsense mutants reveal that the glutamine-binding site of component II is found in the amino-terminal end of the molecule (349).
In Serratia marcescens, the enzymes glutamidotransferase and PRA transferase are specified by separate genes (trpG and trpD, respectively). The DNA sequences of the end of trpG and the beginning of trpD, including the intercistronic region between them, have been compared with the sequences of the corresponding regions of the bifunctional trpD genes of S. typhimurium and E. coli. The intercistronic region is 13 bp long. A hypothetical deletion of one base from this sequence, along with a limited number of base substitutions that remove the stop codon of trpG and the ribosome-binding site of trpD, yields a simple scheme in which a gene like the trpD gene of E. coli or S. typhimurium could be formed by a gene fusion involving the separate trpG and trpD genes of some ancestral strain (247).
Nonsense mutations in trpE can be shown to have a much more dramatic effect on the expression of trpD than on the more distal genes trpC, trpB, and trpA. The basis for this difference was revealed when it was shown that efficient translation of trpD is dependent on efficient translation of the end of trpE (251). A clue to the molecular basis for this translational coupling was discovered when DNA sequence analysis showed that the translation stop signal of trpE overlaps the translation start signal of trpD in the sequence TGATG (247). Although the exact mechanism of translational coupling is not understood, the same phenomenon is observed with the translation of the trpB and trpA polypeptides, in which the same overlapping codons are found (264).
As has been found for the first enzyme in a number of biosynthetic pathways, the activity of anthranilate synthase is inhibited by the end product of the pathway, in this case, tryptophan (140, 160, 185). Tryptophan inhibits both anthranilate synthase activity and PRA transferase activity of the aggregate. Whereas anthranilate synthase can be inhibited 100%, PRA transferase inhibition is incomplete, not exceeding 70%. Uncomplexed PRA transferase is not sensitive to inhibition by tryptophan. The binding of tryptophan and chorismate is competitive, and both sites are postulated to be present on component I. Mutational studies, however, show that the two binding sites are distinct. Conformational changes involving both component I and component II are associated with the binding of substrates or inhibitor and affect anthranilate synthase, glutamidotransferase, and PRA transferase activities (253). A number of mutations which affect either the catalytic or the feedback-sensitive site of anthranilate synthase of S. typhimurium have been identified (51). With these mutated subunits, hybrid complexes that have one catalytically active feedback-insensitive and one catalytically inactive feedback-sensitive subunit with two wild-type PRA transferase subunits have been assembled in vitro. Such a complex is still sensitive to inhibition by tryptophan, indicating the ability of the feedback-sensitive subunit to communicate with and affect the activity of the catalytically active feedback-insensitive subunit present on a separate molecule in the complex (52).
The DNA sequences of the trpE genes of both E. coli and S. typhimurium have been compared. They show a high degree of homology at the amino acid level, with only a 12.5% difference in amino acids. The differences at the DNA level are significantly higher but involve a large number of synonymous codon changes. It is of interest that trpE polypeptides from both organisms do not contain any tryptophan residues (355).
PRA is converted to 1-(o-carboxyphenyl amino)-1-deoxyribulose 5-phosphate (CDRP), and this compound is in turn converted to InG phosphate (InGP) by a single enzyme specified by the trpC gene. PRA and CDRP were first postulated as intermediates in the pathway by Yanofsky (343). CDRP was identified in the dephosphorylated form (CDR) as an accumulation product when certain tryptophan auxotrophs of A. aerogenes and E. coli were starved for tryptophan (111). By using cell extracts of mutants blocked between anthranilate and InGP, Smith and Yanofsky (302) were able to establish that the phosphorylated compound CDRP was an intermediate between anthranilate and InGP. In some mutants unable to form InGP, the conversion of CDRP to InGP was blocked, whereas in others, the blocked reaction was the conversion of anthranilate to CDRP (302). Partial purification of the enzyme from E. coli was achieved in 1960, and the reaction CDRP → InGP was shown to involve only a single enzyme (139). Reactions leading to the formation of CDRP were not studied. Doy et al. (112) extended the work with cell extracts from auxotrophic strains and established that N-(5'-phosphoribosyl) anthranilic acid was an acid-labile intermediate between anthranilate and CDRP. Because of its extreme lability, the conversion of anthranilate to PRA may have been overlooked in previous investigations. The complete purification of the enzyme PRA isomerase-InGP synthetase was achieved in 1966. The enzyme was shown to be a single polypeptide chain with a molecular weight of approximately 45,000, and since the ratio of PRA isomerase activity to InGP synthetase activity was the same in the crude cell extract and the purified enzyme, it was concluded that the two activities reside on the same polypeptide (86). A genetic and biochemical study of missense mutations in the trpC gene established a relationship between the map position of the mutation and the enzymic function lost. Strains with mutations in the proximal half of the trpC cistron (closest to trpD) accumulated CDR; i.e., they lost InGP synthase activity. On the other hand, when the mutations lay in the distal region of trpC (closest to trpB), anthranilic acid accumulated, indicating a loss of PRA isomerase activity. These latter mutants retained InGP synthetase activity, although at a reduced level (301). Limited proteolysis of the purified enzyme has allowed the isolation of large amino-terminal and carboxy-terminal fragments of the enzyme which retain InGP synthetase and PRA isomerase activities, respectively (196). Kinetic studies of the purified enzyme led to the conclusion that the enzyme contained two distinct and nonoverlapping sites for the reaction PRA → CDRP → InGP, and it was proposed that CDRP must dissociate from the enzyme before being converted to InGP. A mixture of mutant enzymes lacking different activities could carry out the overall reaction of converting PRA to InGP. This remains one of the few examples of intracistronic complementation not involving an oligomeric protein (81). It is interesting that in some nonenteric microorganisms, these two activities, PRA isomerase and InGP synthetase, are found in two separate polypeptides (78). Further studies involving a fluorescent analog of CDRP and recent X-ray crystallographic data on the purified enzyme are helping to build up a detailed picture of the reaction mechanisms of this bifunctional enzyme (34, 72, 332). InGP synthetase activity is inhibited by anthranilate (139), and this enzyme is unique among the enzymes of the tryptophan pathway in that it is rapidly inactivated in nongrowing cells of E. coli. This inactivation may involve more than a single mechanism, as both PRA isomerase and InGP synthase activities are not always involved to the same extent, and the kinetics of restoration of activity suggest partial reversibility, whereas studies with antisera suggest that protein degradation or at least denaturation may be taking place (241). Nucleotide sequence studies of E. coli have identified the 1,356 nucleotides of the coding region of trpC. The gene is flanked by intercistronic regions of 11 nucleotides (trpC-trpB) and 6 nucleotides (trpD-trpC) and in this way differs from the trpE-trpD and trpB-trpA junctions (64). Studies of the trpC-trpB intercistronic region in S. typhimurium reveal a 9-nucleotide spacer (294).
Tryptophan synthase was one of the first enzymes of the tryptophan pathway to be extensively studied and has been the subject of a number of review articles (231, 232, 345, 347). It was initially believed that the major function of the enzyme was to convert indole and serine to tryptophan. The enzyme, however, carries out three reactions directly relevant to tryptophan biosynthesis (344, 353):
InGP → indole and d-glyceraldehyde 3-phosphate (1)
indole + l-serine 
l-tryptophan + H2O (2)
InGP + l-serine l-tryptophan + d-glyceraldehyde 3-phosphate + H2O (3)
A consideration of the rates of the three reactions points to reaction 3, namely, the conversion of InGP and serine to tryptophan and glyceraldehyde 3'-phosphate, as the major physiological reaction. This was supported by the demonstration that free indole is not an intermediate in the reaction (80, 82, 343, 353). Early studies showed that two protein components, A and B, were involved in all three reactions (80, 354). Subsequent work revealed that tryptophan synthase is a tetramer of two α units specified by the trpA gene and a β 2 dimer specified by the trpB gene. The β 2 subunit has two binding sites for pyridoxal phosphate and two independent sites to which α units can bind (85, 334). The α subunit has the ability to convert InGP to indole and glyceraldehyde 3'-phosphate, and the β 2 subunit is able to convert indole and serine in the presence of pyridoxal phosphate to tryptophan. In both cases, however, the α 2 β 2 complex carries out these reactions at greatly increased rates (27, 80). The α subunit has been purified and crystallized and has a molecular weight of 29,000 (166, 197, 292). The amino acid sequence of the α subunit has been determined (215, 348). As is the case for the trpE-encoded polypeptide, the α subunit does not contain any tryptophan residues. The analysis of the amino acid substitutions that occurred in certain trpA mutants and the map location of the mutations causing these changes provided the first compelling evidence of colinearity between the gene and the polypeptide encoded by it (348).
The β 2 subunit has also been purified and crystallized. It has a molecular weight of 89,000 (1, 2, 142, 158, 159, 233). The β 2 subunit and the α 2 β 2 complex catalyze other reactions involving pyridoxal phosphate, but these reactions are not directly relevant to aromatic biosynthesis (232). The binding of pyridoxal phosphate and the interaction of the subunits has been extensively studied (25, 85, 170, 232, 316, 317). The native α 2 β 2 complex (molecular weight, 147,000) has been purified and crystallized from E. coli and shown to be identical with the reconstituted tryptophan synthase complex (1, 151).
Tryptophan synthase has been used to investigate the evolutionary relationship between E. coli and S. typhimurium. Because tryptophan synthase is a mutienzyme complex, experiments were designed to test whether single components from S. typhimurium could replace corresponding components in the E. coli complex without destroying activity. A number of experiments involving in vitro and in vivo construction of hybrid complexes confirmed that such substitutions could occur (18, 83, 84, 244).
A comparison of the nucleotide sequences of the trpA and trpB genes in E. coli and in S. typhimurium reveals a very close similarity between the polypeptides synthesized by both organisms (79, 248). For the trpB-encoded polypeptide, more than 96% of the amino acid sequences are identical, and for that of trpA, about 85% are identical. Studies of the nucleotide sequence of the mRNA corresponding to the intercistronic region show that in both organisms, the end of the trpB gene and the beginning of the trpA gene overlap in a sequence at the mRNA level of UGAUG. An RNA fragment of 40 nucleotides containing this central AUG codon is protected from nuclease attack by ribosomes and contains an identifiable ribosome-binding site (264). As with trpE and trpD, translational coupling occurs between trpB and trpA (6).
The tryptophan synthase complex has also been crystallized from S. typhimurium (4). Resolution of this structure to 2.5 Å (0.25 nm) has been achieved, and a detailed three-dimensional structure has been proposed (179). An essential feature of this structure is a hydrophobic tunnel of about 25 Å (2.5 nm) linking the active sites of the α and β subunits. The diameter of the tunnel matches that of the intermediate substrate indole, and it has been proposed that this tunnel facilitates the diffusion of indole from the active site in the α subunit to the active site in the β subunit. It has also been proposed that the tunnel prevents the escape of indole to the solvent during catalysis. Site-specific mutagenesis is being used to probe this proposed structure and to investigate the mechanisms involved in the interactions between active sites in the α and β subunits (5, 45, 119, 341).
The chromosomal locations of the structural genes for the enzymes of the pathway of biosynthesis of the aromatic amino acids, the transport systems, and the pathway’s two major regulator genes are shown in Table 2. Apart from the fact that the trp operon genes lie on an inverted segment of chromosome in S. typhimurium, the overall distribution of genes is the same. In some cases, a gene may be present on one map but not on the other, e.g., tyrP, aroLM, and pheP. It is likely that these genes will be found in the same location in the other organism, but this has not yet been established. The function of the gene aroM is not known. However, with aroL and an additional open reading frame, it makes up a single transcription unit that is under the control of the tyrR gene product (104).
Table 2Genes for biosynthesis, activitation, and transport of aromatic amino acids |
The isolation of mutants and the mapping of most of the structural genes have been straightforward. Gene designations were independently allocated to five of the genes for the common pathway in S. typhimurium and E. coli (146, 249, 261). Unfortunately, not all of the gene designations coincided, but this was remedied in more recent maps (16, 282). The cloning of most of these genes in E. coli has provided detailed molecular information on their map locations, with particular reference to Kohara’s physical map of the chromosome (202), and this is also shown in Table 2.
Feedback inhibition of various key enzymes of the pathway by the aromatic amino acids has already been discussed. In this section, the various controls on gene expression and the ways in which these controls have been investigated are considered. As far as we know, all of the controls affect the transcription of these genes rather than the translation of their mRNAs. Specific regulation of transcription is achieved by repression or activation, and in the case of the trp operon (187, 346), the pheA gene, and the pheST genes (129, 130, 131, 147, 174, 226, 360), some regulation is achieved by attenuation. In the case of aroF, there is an as yet undefined control in addition to repression (53). Metabolic regulation, as described by Rose and Yanofsky (275), exerts a general control on the aromatic pathway (314) and on the genes for the aminoacyl-tRNA synthetases (246). As Table 2 shows, there are only four examples of tightly clustered genes on the map, namely, the trp operon (trpEDCBA), aroF tyrA pheA, aroK aroB, and aroLM. The genes trpEDCBA, aroF tyrA, and aroLM each make up a classic operon. The genes aroF tyrA and aroLM are part of the TyrR regulon, and aroLM is also part of the TrpR regulon, as is trpEDCBA. The genes aroK and aroB are part of a single transcription unit but do not appear to be subject to specific regulation (217). The genes controlled by either or both of the regulator proteins TyrR and TrpR are listed in Table 3.
Table 3Modulation of gene expression by TyrR or TrpR or both |
Five separate transcriptional units are currently identified as belonging to the TrpR regulon. The first of these is the classic trpEDCBA operon (32, 352). The tight linkage of the genes of the trp operon to each other was established in some of the first mapping experiments to be carried out in E. coli with the generalized transducing phage P1 (351). A similar tight linkage was established in Salmonella spp. (35). The subsequent discovery that these genes could be picked up in E. coli by the specialized transducing phage φ?80 (102, 222) or incorporated into phage λ (274) coupled with the generation of deletions which extended from the region containing tonB into the trp operon resulted in the in vivo cloning of these genes. This permitted their use in in vitro studies considerably in advance of the development of in vitro cloning methods. Other members of the regulon are aroH (150), the trpR gene itself (152), aroL (163, 208), and mtr (161, 286). The TrpR-binding sites or operators for each of these transcription units are shown in Fig. 5.
Each TrpR-binding site differs significantly from the others in its positioning within or, in the case of aroL, outside of the promoter region. Klig et al. (198) suggested that the variation in repression from 300-fold for the trp operon to 6-fold for aroH and 3-fold for trpR is a direct consequence of the relative positions of the trpR binding sites and their respective promoters. They report finding no difference in affinity between the TrpR protein and these three operators in a number of in vitro and in vivo tests. In separate studies, Kumamoto et al. (204) reported in vitro DNase and methylation protection studies involving the TrpR protein and the operators of the trp operon (henceforth referred to as trp), of trpR, and of aroH. From their results, they concluded that the trp operator binds three TrpR dimers in tandem, the aroH operator binds two, and the trpR operator binds one. By analogy with other systems, the number of repressor molecules bound would also be expected to influence the degree of repression. On the other hand, in more recent studies, Liu and Matthews (216) reported that gel retardation studies indicate that the trp operator and the aroH operator each bind two TrpR dimers. Furthermore, they reported that the affinity between the TrpR protein and the trpR operator is 1/10 of the affinity between the TrpR protein and the trp operator. The apparent differences with previous reports have yet to be resolved.
The very unusual position of the TrpR-binding site in aroL is explained by the finding that in this system, TrpR binding enhances only TyrR-mediated repression, which occurs when TyrR protein binds at sites further upstream and overlapping the RNA polymerase-binding site. The TrpR enhancement may also involve an interaction between the TrpR and TyrR proteins (163, 208).
The trp operator was first identified by the isolation and characterization of various operator constitutive mutants (32). The point mutants that were isolated had base pair changes at positions –6 and –5 and +5 and +6 (Fig. 5). In an extensive study that used the challenge phage system, Bass et al. (27, 28) made symmetrical base substitutions at 11 positions in the trp operator. Mutations affecting CTAG at positions –6–5–4–3 and +3+4+5+6 reveal the importance of this sequence in operator function. Similar results were obtained by Staacke et al. (310).
In a separate study, Czernik et al. (91) used a Trp repressor affinity column to select from a randomized pool of synthetic double-stranded DNA those sequences specifically bound by the column in the presence of tryptophan and eluted on the addition of the tryptophan analog β-indole acrylic acid. Analysis of these sequences shows that CTAG is the motif that is critical for binding to repressor.
The trpR gene was one of the first regulator genes to be identified and mapped (71). It was cloned independently by Gunsalus et al. (153) and Roeder and Somerville (272), and its sequence was determined (152). From the sequence, the translation product of the gene was deduced to be a polypeptide of 108 amino acids (152). The protein was shown to exist as a dimer (189). A mutational analysis carried out by Kelley and Yanofsky (194) to look for negative dominant mutations identified a putative helix-turn-helix motif (HTH) toward the carboxyl end of the protein, which contained amino acids essential for repressor action (194). Further analyses of the HTH identified key amino acids and led to predictions of possible interactions between certain amino acids and particular nucleotides in the trp operator (27, 254).
The TrpR protein was crystallized, and its structure was determined to a resolution of 2.6 Å (0.26 nm) (287) and subsequently to 1.8 Å (0.18 nm) (358). It is a mainly helical structure. Each monomer comprises six helices (A through F), and the dimeric protein has a structure in which five of the six helices are interlinked (236). Helices D and E make up the HTH, and the first seven amino acids from the amino-terminal end of the protein appear to represent a flexible terminal arm. Two molecules of tryptophan are bound per dimer, and the binding of tryptophan alters the critical orientation of the HTH substructure essential for operator-specific recognition (221). When tryptophan binds to the apo repressor, the two E helices of the dimer move 5 to 9 Å (0.5 to 0.9 nm) apart so that they fit into adjacent major grooves of the DNA (219, 358). Some of the various studies of the Trp repressor have been reviewed elsewhere (303).
Otwinowski et al. (252) cocrystallized the Trp repressor and a 19-bp operator 
and resolved its structure to 2.4 Å (0.24 nm). Unexpectedly, there were no direct hydrogen bonds between functional groups of the bases and the amino acids of the repressor. Instead, the interactions were mediated by a layer of water molecules at the interface between protein and DNA. A process of indirect readout in which the repressor recognized sequence-dependent structural variations unique to the trp operator was proposed. This interpretation is strongly argued in a review by Luisi and Sigler (219), and support for structural changes in the DNA when the repressor binds is provided by nuclear magnetic resonance studies of solution structures of trp-repressor-operator DNA complexes (357).
Staacke et al. (310) proposed that the DNA sequence used in these structural studies does not represent the true operator, which, they suggested, involves a larger sequence to which two dimers bind with centers between –4 and –5 and +4 and +5 (so-called β symmetries). Further experimentation and careful analyses, however, have ruled out this possibility (58, 157). Controversy about the role of water molecules at the protein-DNA interface, however, still continues. Brennan and Matthews (39) and Carey et al. (58) suggested that the conditions required for crystal growth may favor nonspecific binding of the type observed with an interface of water molecules. Furthermore, on the basis of the altered DNA-binding specificities of two TrpR mutants which have the amino acid serine or lysine substituted for threonine at the third position of the second helix of the HTH, Pfau et al. (254) argue for direct specific contacts between the amino acids in the TrpR repressor and the trp operator DNA. Also, nuclear magnetic resonance solution structures of the repressor-operator complex as reported by Zhang et al. (357) do not include a layer of water molecules at the protein-DNA interface. A generally agreed upon position on the interaction between TrpR protein and its operator targets has therefore not yet been reached.
The number of molecules of Trp repressor that bind to the trp operator in vivo is also not resolved. In vitro studies suggest either three or two dimers per trp operator target when a 40-bp target is used (204, 216) and one dimer if a 20-bp operator is used (58, 216). If, on the other hand, a 16-bp palindromic DNA containing a central trp operator half-site is complexed with the TrpR repressor, paradoxically, the stoichiometry of the complex is found to be 2:1, with two dimers binding tandemly to the half-site (209). In this same study, it was shown that the N-terminal arms of the repressor increase the binding affinities of tandemly bound dimers, probably by mediating dimer-dimer interactions. Such binding to half sites could be of particular relevance to aroL and mtr, in both of which the right-hand trp half-sites appear to be very weak.
The TyrR regulon comprises eight or possibly nine separate transcription units (40, 44, 54, 55, 104, 122, 143, 145, 161, 180, 181, 182, 223, 242, 286, 322, 330, 340). TyrR protein can act as a repressor and, in some cases, as an activator. Its major coeffector is tyrosine, although phenylalanine and tryptophan also modulate its activity. The next section summarizes what is currently known about this protein and its various activities.
The role of the TyrR protein in repression and activation has been reviewed elsewhere (257). The tyrR gene (322) has been cloned, and its sequence has been determined (69, 75, 76, 339). Judging from the sequence, the TyrR protein comprises 513 amino acids with a calculated subunit molecular weight of 57,640, compared with a measured molecular weight of 110,000 ± 5,000 for the homodimer, in which form the protein normally occurs in the absence of effectors (12, 89).
The protein has been overexpressed and purified in a number of laboratories (10, 12, 89). Limited proteolysis indicates that the protein has two or possibly three domains. The amino-terminal domain involves residues 1 through 190, the central domain is residues 191 through 467, and the putative third domain is residues 468 through 513 (89) (see Fig. 6).
Each of the domains is important for a different function of the TyrR protein. These functions are briefly discussed.
The Amino-Terminal Domain.
Deletions and specific amino acid substitutions have identified the region of the protein between amino acids 2 and 19 with a critical role in activation of the expression of tyrP and mtr. A number of mutations affecting this region abolish TyrR-mediated activation without affecting repression of the other members of the regulon (88, 90, 338, 339). A series of small deletions extending further into the protein and subsequent site-directed mutagenesis have identified a second region between amino acids 92 and 103 which may play a subsidiary role in activation (J. Yang, H. Camakaris, and A. J. Pittard, Proc. ASBMB 25:-2-6, 1993). Recent in vitro transcription studies with the tyrP promoter indicate an interaction between TyrR protein and RNA polymerase as the likely basis for TyrR-mediated activation (B. Lawley, A. Ishihama, and A. J. Pittard, Proc. ASBMB 25:146, abstr. POS-2-6, 1993). In such in vitro systems, a twofold activation of transcription that is comparable to that observed in vivo can be demonstrated when both phenylalanine and TyrR protein are present. This activation does not occur, however, if the RNA polymerase lacks 73 amino acids from the carboxyl end of its α subunit. Such mutant polymerases fail to be activated by a number of activator proteins such as cAMP receptor protein and OmpR, which are referred to as class I activators (183). On the basis of these results, the most likely hypothesis is that TyrR-mediated activation of both tyrP and mtr expression involves a specific interaction between the amino-terminal region of TyrR and the carboxyl-terminal region of the α subunit of the RNA polymerase.
The Central Domain.
The central domain of TyrR is homologous to the central domains of a number of activator proteins, including NtrC, NifA, DctD, XylR, and FhlA, which activate expression from σ 54 promoters (discussed in reference 257). Mutational studies of the NtrC protein showed that the central domain of this protein is essential for activation (14, 325). A key component of this central domain is an adenylate-type ATP-binding site. Mutations that cause amino acid substitutions in site A of the ATP-binding site and a number of other mutations in this central domain destroy the ability of NtrC protein to activate gene expression. Furthermore, in the case of NtrC, ATP hydrolysis is involved in converting the RNA polymerase- promoter complex from a closed to an open state (325). The situation with regard to TyrR is quite different. Mutations altering site A of the ATP-binding site and three other mutations affecting the highly conserved residues E-274, G-285, and E-302 of the central domain have no effect on activation but abolish TyrR-tyrosine-mediated repression of aroF, aroL, tyrP, and tyrB. They have a partial effect on aroP expression but no effect on TyrR-mediated repression of aroG (339; J. Yang, unpublished data). Furthermore, a study of truncated TyrR proteins indicates that the region between residues 194 and 438 is critical for dimerization (90). It has been demonstrated (12) that purified TyrR protein binds ATP with a Kd of 5 to 7 μM. In the presence of excess ATP, TyrR protein binds tyrosine with a Kd of 50 μM. Tyrosine binding cannot be detected in the absence of ATP. It has recently been shown that in the presence of the nonhydrolyzable ATP analog ATP?S and tyrosine (500 μM) or phenylalanine (25 mM), TyrR protein self-associates to form a hexamer (336). This hexameric form is postulated to play a major role in tyrosine-mediated repression and presumably cannot be formed in these central-domain mutants of TyrR. The role of ATP hydrolysis in repression has not yet been resolved. The purified TyrR protein does have ATPase activity that is stimulated by tyrosine (87, 336). Cui et al. (87) estimate that the level of this activity is similar to that reported for the unphosphorylated form of the NtrC protein. Weiss et al. (325) reported that activation of NtrC by phosphorylation increases its ATPase activity about 11-fold. Although tyrR mutants with amino acid substitutions in site A of the ATP binding site have been isolated and shown to have lost the ability to carry out TyrR-tyrosine-mediated repression (339), these mutant proteins have not yet been purified and tested for ATP binding and hydrolysis. The finding by Wilson et al. (336) that the nonhydrolyzable ATP analog ATPγS can substitute for ATP in the self-association of TyrR dimers into hexamers might indicate that only binding is required for repression. If this is the case, perhaps ATP hydrolysis functions to facilitate the reverse reaction of hexamer to dimers, hence freeing operator sites. It should, however, also be noted that in the case of NtrC protein, Austin and Dixon (13) reported that ATPase activity is stimulated by DNA containing NtrC-specific binding sites, and Wilson et al. (336) hypothesized that hexamerization might normally take place while TyrR protein is associated with the DNA.
The Carboxyl Domain.
The carboxyl domain contains a classic Cro-like HTH DNA binding motif. Amino acid substitutions of a number of residues within this region destroy the ability of the TyrR protein to repress the various genes of the regulon (339). Similarly, a truncated TyrR protein missing the HTH motif can neither repress nor activate members of the regulon (90). Deletion of 10 of the 11 amino acids between helix 2 and the carboxyl terminus also destroys activation and repression functions (339). A representation of the TyrR protein showing the three domains and the critical amino acids that have been discussed in this section is given in Fig. 6, and a discussion of the DNA sites to which the protein binds is included in the next section.
The target sites on the DNA that are recognized by the TyrR protein are all related to the palindromic sequence TGTAAAN6TTTACA. The underlined bases (G and C) are invariant, and changing them to any other base largely destroys the function of the box (10, 30, 66, 68, 128, 192, 208, 283, 286, 340; B. Lawley and S. Hwang, unpublished data). These palindromic sequences, together with the two flanking nucleotides at each end, are referred to as TyrR boxes. Their distribution with regard to the putative RNA polymerase-binding sites for each of the transcription units is shown in Fig. 7 along with relevant TrpR-binding sites for aroL and mtr. The sequence of each box is shown in Fig. 8. All of the transcription units repressed by tyrosine, i.e., aroF, tyrA, aroL, aroP, tyrB, and tyrP, have two adjacent TyrR boxes separated by a single base. In every case, one of these boxes shows a closer agreement to the consensus sequence than the other and in in vitro experiments is bound by the TyrR protein in the absence of effectors (9, 340; B. Lawley, unpublished data; V. S. Argyropoulos, Ph.D. thesis, University of Melbourne, Melbourne, Australia, 1989). For convenience, these boxes are referred to as "strong" boxes. Strong boxes, with the exception of aroG, are also AT rich in the central six positions (Fig. 8). The adjacent "weak" boxes are bound by TyrR protein only in the presence of tyrosine and ATP, have a weaker agreement with the consensus, and contain two or more G’s and C’s within the central six bases. With the exception of aroP, in which the boxes lie downstream of the RNA polymerase-binding site, the weak box always overlaps the RNA polymerase-binding site. In order for the weak box to be bound by TyrR, a strong box must be present nearby on the same face of the DNA helix. In the case of tyrP, this cooperative binding between strong and weak boxes still occurs if the boxes are separated by one turn of the helix but not if the separation involves three turns of the helix (9).
It has been proposed that cooperative binding to two or more boxes involves the hexameric form of the protein formed in the presence of tyrosine and ATP (336).
One exception to the general rule that the weak box plays a major role in tyrosine-mediated repression is the recent finding that box 2 of aroL apparently plays no part in repression. Like the other weak boxes, it is bound by TyrR protein only in the presence of tyrosine and ATP, but mutating the invariant G-C pairs in this box has no effect on repression, which appears to involve a TyrR-mediated loop between strong boxes 1 and 3, which are separated by 54 bp (208). The aroF tyrA operon also has a single strong box separated by three turns of the helix from an adjacent strong-box–weak box combination, and DNA looping between the strong boxes is again a possibility. In this case, however, the order and position of the boxes are different from those in aroL (Fig. 7). In aroL, inactivation of either box 1 or box 3 results in complete derepression (208), whereas in aroF, although inactivation of box 2 reduces repression to less than 2-fold, inactivation of box 3 only partially reduces repression, from 150-fold to 25-fold (68). By inserting DNA between boxes 2 and 3 of aroF, Cobbett (67) demonstrated that the involvement of box 3 in repression is virtually unaltered if the distance between the boxes is increased to 110 bp and that such involvement is still evident though reduced when the distance is increased to 400 bp. If the box is moved any further upstream, it no longer contributes to repression. Although in the wild type box 3 is on the same face of the helix as box 2, results with spacing mutants suggest that this arrangement is not essential for these interactions between two strong boxes with intervening distances of 50 bp or more (67).
None of the genes of the TyrR regulon have two strong boxes adjacent to each other. When such a situation is engineered by changing the palindomic arms of weak box 2 of aroL so that they agree with the consensus sequence TGTAAAN6TTTACA, the TyrR-tyrosine-mediated repression of aroL seen in the wild-type arrangement is replaced by a TyrR-mediated tyrosine-independent repression of equivalent strength (208). Similar results have been obtained by creating two adjacent strong boxes for aroG (N. Bassegio, Ph.D. thesis, University of Melbourne, Melbourne, Australia, 1992). We assume that two adjacent strong TyrR boxes can be effectively bound by TyrR dimers in the absence of tyrosine and that the molecular basis for tyrosine-mediated repression in the wild type is an arrangement of TyrR boxes that, because of the location of the boxes and the relative strength with which each is bound by the TyrR protein, requires the hexameric form of the protein for effective repression to occur. Such situations occur when a strong and a weak box are located on the same face of the helix immediately adjacent to each other, as in the case of tyrP, tyrB, aroF, and aroL, or are no more than one turn of the helix apart (9) or when two strong boxes are located further apart but need to be involved in a TyrR-mediated DNA loop formation for repression to occur, as is probably the case with aroF and aroL.
The level of repression in minimal medium varies for each of the genes of the regulon. Some examples are shown in Table 4. The basis for this repression in minimal medium has always been uncertain. Is it driven by the endogenous pools of amino acids in the cell, or does it represent transcriptional control exerted when TyrR dimers without coeffectors bind to strong TyrR boxes? A recent observation (J. Yang, unpublished data) that the levels of expression of aroL, aroF, tyrP, and tyrB in tyrR cells with the tyrR E274Q allele on a multicopy plasmid are approximately the same as the levels seen in a strain with the tyrR + allele on a multicopy plasmid when both are grown in minimal medium might argue for the latter explanation. The E274Q mutant is unable to carry out any tyrosine-mediated repression, and the minimal medium repression seen with this mutant presumably results from uncomplexed TyrR dimers. The different level of minimal medium repression for each gene presumably reflects the different affinities between the TyrR dimers and the various strong boxes and the different extents to which TyrR dimers bound to these boxes can interfere with transcription.
Table 4Specific activities of various proteins of the tyrR regulon |
The relative positions of strong boxes upstream of the RNA polymerase-binding site are also critical for activation. By changing the positions of such boxes, it has been established that for activation, the optimal position of the center of the TyrR box is 32 or 42 bp upstream from the center of the –35 sequence (9, 283).
In the case of aroP, both of the adjacent TyrR boxes lie downstream of the RNA polymerase-binding site. Mutational studies (66; Lawley, unpublished data) and in vitro protection experiments confirm the role of these boxes in repression (Lawley, unpublished data). It has not been established whether binding of TyrR protein to these boxes stops transcription elongation by the roadblock mechanism proposed for purB (160), but on the basis of available evidence, this seems to be the most likely mechanism. The regulation of aroP also differs from that of the other tyrosine-regulated genes in that its repression can be caused by tyrosine, phenylalanine, or tryptophan and its repression is largely unaffected by the E274Q mutation.
The single TyrR box in aroG is unusual in that it combines a GC-rich central six bases with two perfect palindromic arms. Site-directed mutagenesis and in vitro protection studies confirm the role of this box in the regulation of aroG (29). In in vitro experiments, the affinity of the box for TyrR protein is significantly less than that of an equivalent strong box of tyrP with an AT-rich central region (10, 29). Although in vivo studies have revealed that phenylalanine and tryptophan play important roles in the repression of wild-type aroG (44, 180), no similar effect was seen in the expression of the cloned promoter-operator region of aroG (29). Further studies are required to resolve these different results.
The expression of mtr, the gene encoding the tryptophan-specific high-affinity transport system, and aroL, the gene for shikimate kinase II, is modulated by both tryptophan and tyrosine. Expression of mtr is repressed by tryptophan, and in the absence of tryptophan, it is activated by tyrosine or phenylalanine. The tryptophan repression requires a functional TrpR protein, and the tyrosine and phenylalanine activation is mediated via the TyrR protein. The involvement of both these systems has been verified by studying expression in trpR and tyrR mutants (161) by in vitro protection experiments and site-directed mutagenesis of the relevant TrpR and TyrR boxes (283, 286). A possible interaction between TrpR and TyrR proteins has been proposed to explain different levels of activation observed in trpR + and trpR strains (283).
In the case of aroL, tryptophan enhances tyrosine-mediated repression. The tryptophan effect requires a functional TrpR protein and is totally dependent on the action of TyrR protein. No effect due to tryptophan or TrpR protein is seen in tyrR strains (163). Again, TrpR and TyrR boxes have been identified, and their functions have been verified by site-directed mutagenesis and in vitro protection experiments with purified proteins. If the sequence is manipulated so that the TrpR box is separated from the nearest TyrR box by half a turn of the helix, tryptophan enhancement of tyrosine repression is largely abolished. This result also implies a possible interaction between TyrR and TrpR proteins (208).
It has been reported that the expression of aroH, which is part of the TrpR regulon, is also modulated by TyrR (242). In this case, however, no TyrR-binding sites have been identified, and site-directed mutagensis of any potential TyrR-binding sites has failed to influence expression of the gene (J. Sarsero, unpublished data).
The expression of aroP, the gene for the common aromatic amino acid transport system, is also repressed by phenylalanine, tyrosine, and, to a lesser extent, tryptophan. In this case, however, mutating the trpR gene has no effect on repression, and no functional TrpR-binding sites have been identified (B. Lawley, unpublished data).
Whether cells of E. coli are grown in minimal medium or in minimal medium supplemented with the aromatic amino acids, the level of expression of the pheA gene is the same. However, starving cells for phenylalanine or introducing specific mutations can result in a 2- to 20-fold increase in the level of pheA expression (44, 148, 181). Strains of E. coli derepressed for synthesis of the pheA gene product, chorismate mutase-prephenate dehydratase, were found among mutants resistant to 3- or 4-fluorophenylalanine. Genetic studies showed that the mutations in these strains are tightly linked to the pheA structural gene and are not recessive in diploids. On this basis, such mutations were provisionally identified as mutations in a putative operator locus of the pheA gene (180). Attempts to isolate strains with mutations in a putative regulator gene, pheR, using the same selection were not successful (S. W. K. Im and A. J. Pittard, unpublished data). In S. typhimurium, on the other hand, the first mutants to be isolated that were derepressed for chorismate mutase-prephenate dehydratase were not operator mutants but mutants with changes in a gene (pheR) that was unlinked to the pheA structural gene. These mutations were recessive in partial diploids that were derived by using the F-merogenote F116 from E. coli. This F-merogenote contains the 59- to 65-min region of the E. coli chromosome. Crosses with Salmonella Hfr donors had shown that pheR + was located in this same region of the Salmonella chromosome (144).
Putative pheR mutants were ultimately isolated in E. coli after the construction of pheA-lacZ operon fusion strains and selection for mutants able to grow on media containing phenyl-β-d-galactoside as sole carbon source (147, 148, 149). These mutant strains were derepressed for the synthesis of chorismate mutase-prephenate dehydratase and, as was the case in S. typhimurium, were converted to a repressible state upon the introduction of the F-merogenote F116. Insertional mutagenesis appeared to implicate a 14-kDa protein as the pheR product. On the basis of these results, pheR was proposed to encode a putative repressor protein (148). However, sequence analysis of the pheR gene followed by detailed mutational analyses showed that the gene termed pheR encoded a tRNAPhe molecule (129, 131). When the putative operator mutants of pheA were also cloned and the sequences were determined, these mutants were shown to have changes in the stem of the transcription terminator of the pheA leader sequence (130, 359). Although a number of reports taken together lead to the conclusion that there are three or possibly four structural genes for tRNAPhe in E. coli (48, 49, 127, 129, 149, 293, 335), there are, in fact, only two (260). The reports of pheR, pheU, and pheW all refer to the same gene near min 94.5 on the E. coli map which should be referred to as pheU. The gene pheV, which is the second gene for tRNAPhe, is located at map position min 64 (47). Apparently, a mutation in either pheV or pheU reduces the level of tRNAPhe in the cell to a point at which attenuation of both pheA expression and pheST expression is relieved, resulting in increased expression of these transcription units. This increased expression is reversed when the levels of tRNAPhe are increased by introducing either pheU or pheV on a multicopy plasmid. Consequently, it is now understood that pheA expression is controlled solely by attenuation and does not normally involve any repression. This conclusion is also supported by the work of Borg-Olivier et al. (38), who reported the isolation of mutants of E. coli resistant to the phenylalanine analog p-fluorophenylalanine and derepressed for pheA expression as a result of a mutation which mapped to the pheST genes which encode the phenylalanyl-tRNA synthetase. The observation by Bogosian and Somerville (36) that pheA transcription was repressed 10- to 14-fold in strains that overproduce the TrpR protein remains unexplained but appears to involve an unusual event resulting from the very high levels of TrpR protein.
Tryptophanyl-tRNA synthetase is an α 2 homodimer with a measured subunit molecular weight of 37,000 (190, 243). The gene was cloned, and its sequence was determined. The protein has an estimated M r of 37,428 (154). Transcriptional trpS-lacZ fusions have been constructed and used to investigate whether trpS expression is regulated in any specific way. As is the case for genes for other aminoacyl-tRNA synthetases, trpS expression is subject to metabolic regulation, and expression increases 2.5-fold when the doubling time is decreased from 150 to 37 min. Starvation for tryptophan or the introduction of a trpR mutation or a mutation in miaA, which affects attenuation of the trp operon, has no effect on trpS expression (155). On the other hand, mutations in the trpS gene do affect attenuation control of the trp operon and, as previously mentioned, enhance aroF expression in tyrR cells (53).
Tyrosyl-tRNA synthetase is an α 2 homodimer. The molecular weight of the subunits was determined by Calender and Berg (50) and Chousterman and Chaperville (63), who gave weights of 47,500 and 48,000, respectively. The gene was cloned, and its sequence was determined. The translated product of 423 amino acids had an estimated M r of 47,403 (24). Overexpression of tyrosyl-tRNA synthetase to levels of about 10% of total cell protein has been successfully used in its purification (23). Increased expression to above 55% of cell protein is very toxic to cells. The toxicity is a consequence of the increased synthetase activity almost certainly causing misincorporation of amino acids into proteins (31). There have been few reported studies on the regulation of this enzyme, which, however, is presumed, like the other synthetases, to be subject to metabolic regulation. The gene for pyridoxine 5'-phosphate oxidase, pdxH, lies immediately upstream of tyrS. There is no detectable transcription terminator between the two genes, and 20% of the tyrS transcripts originate from the pdxH promoter (207).
Phenylalanyl-tRNA synthetase has a more complex structure of the type α 2 β 2. Equilibrium sedimentation studies give an overall M r of 240,000, which is consistent with an α 2 β 2 structure in which the α subunit has an M r of 37,000 and the β subunit has an M r of 98,000 (123). The α subunit is encoded by the pheS gene, and the β subunit is encoded by the pheT gene (74).
The pheS and pheT genes form a single transcription unit and are transcribed from a single promoter upstream of pheS (265). Such a unit provides one of many examples defined as operons that contain no sensu stricto operators. The nucleotide sequences of the pheS and pheT genes and flanking regions have been analyzed and reveal a leader sequence preceding pheS that has all the features of a classic attenuator. It includes a small open reading frame of 14 amino acids, of which 5 are phenylalanine, and an RNA able to form alternate structures, one form of which results in premature transcription termination (124, 229). The control of pheST expression by attenuation has been confirmed with lacZ fusions, by alteration of the level of aminoacylated tRNAPhe (305), and by site-directed and deletion mutagenesis of the leader region (226, 304). In those studies (304), it was also established that 30% of the pheST transcripts result from readthrough from the promoter of the upstream rplT gene, which encodes the ribosomal L20 protein. The estimated M r of phenylalanyl-tRNA synthetase as calculated from the DNA sequence data is 247,562 (229), which is in good agreement with the value of 240,000 determined by equilibrium centrifugation. Phenylalanyl-tRNA synthetase was among the first synthetases to have its expression shown to be subject to metabolic regulation (246). Temperature-sensitive mutants of pheS or pheT show greatly increased synthesis of chorismate mutase-prephenate dehydratase as a consequence of deattenuation of pheA expression (38). Such temperature-sensitive strains have also been used to select genes for tRNAPhe, which, when present in increased copy number, can suppress the ts mutation in the synthetase (48, 293).
Early studies with S. typhimurium (7) and E. coli (41) established that each organism possessed one transport system which could transport all three of the aromatic amino acids. This system was termed the general aromatic transport system and was shown to be encoded by the gene aroP (8, 41). In addition to the general transport system, both organisms contained three specific transport systems able to transport tyrosine, phenylalanine, or tryptophan. The Km for individual substrates in each of these specific systems (2 × 10–6 M) was approximately 10 times greater than the Km of the general transport system (4 × 10–7 M) for its substrates (41). Studies of the catabolism of tryptophan identified an additional tryptophan transporter formed when tryptophan was being used as a source of carbon; this transporter was referred to as a high-capacity, low-affinity system (120) and had a Km for tryptophan of 10–5 M.
Mutants lacking each one of these systems have been obtained, the respective genes have been mapped and cloned, and their sequences have been determined (41, 65, 162, 172, 257, 285, 329). In each case, the protein predicted from the sequence is very hydrophobic, with the general characteristics of polytopic integral membrane proteins such as lactose permease (191). On the basis of amino acid sequence homologies, the two tryptophan-specific proteins Mtr and TnaB and the tyrosine-specific protein TyrP can be grouped into a single family. Mtr and TnaB show 52% homology to each other and about 32% homology to TyrP. Consideration of hydropathy profiles, distribution of charged residues, and incidence of proposed β turns led to a model for Mtr of a polytopic protein with 11 spans across the membrane, with the amino terminus in the cytoplasm and the carboxyl terminus in the periplasm (285). The essential features of this model have recently been confirmed by the construction of numerous Mtr-PhoA and Mtr-LacZ fusion proteins (283, 284). No other proteins in the database show homologies to these three. On the other hand, AroP and PheP proteins are part of a large family of membrane proteins involved in the transport of amino acids, polyamines, and choline (270). Some of these amino acid transporters, such as LysP and GabP and the putative transporter YifK, are from E. coli, whereas others, such as Can1, Gap1, Put4, YCC5, and Hip1, are from yeast cells. The amino acid sequences of PheP and AroP show 60% homology. A topological model for PheP involving 12 membrane spans was proposed (255) and has recently been confirmed in broad outline by studies of PheP-PhoA fusions (J. Pi, unpublished data). There has also been extensive site-directed mutagenesis of pheP and the identification of a small number of charged amino acids located within the membrane and essential for transport activity (256). In the case of each of these five systems, studies with uncouplers indicated that transport is energized by the proton motive force (337; M. Whipp, Ph.D. thesis, University of Melbourne, Melbourne, Australia, 1977; Sarsero, unpublished data).
A recent unexpected finding is that when expression of pheP is increased, either by changing its promoter or by increasing its copy number, the PheP system can be shown to transport tyrosine as well as phenylalanine. The Km for tyrosine is 10 times greater than that for phenylalanine, so that in a sense, the transporter is still phenylalanine specific (A. Cosgriff and A. J. Pittard, unpublished data).
An unresolved feature of aromatic transport is the role played by the azaB gene. It has been reported (333) that mutations in this gene impair the active transport of phenylalanine in aroP mutant cells. The azaB gene maps in a different location from the pheP gene, and its function is unknown. The wild-type gene has not yet been cloned, nor has its sequence been analyzed.
As previously mentioned, those intermediates of the pathway that are phosphorylated are not transported into the cell. Nutritional studies of aromatic auxotrophs indicated that the intermediates that appeared to be taken up by the cell were DHQ, dehydroshikimate, and shikimate from the common pathway and anthranilate and indole from the tryptophan pathway. Although hydroxyphenylpyruvate and phenylpyruvate from the tyrosine and phenylalanine pathways are not normally taken up by cells, mutants that are able to utilize these compounds can be isolated (133). Very little work has been done with these various transport systems. What little has been done is reported below.
Indole.
In a system in which the tryptophan requirement of a tryptophan synthetase mutant was provided by the enzyme tryptophanase converting exogenously provided indole into tryptophan, Yanofsky et al. (350) established that the Mtr transporter was required to transport indole into the cell.
Anthranilate.
No studies concerning the transport of anthranilic acid have been reported.
Phenylpyruvate and 4-Hydroxyphenylpruvate.
Although Gelfand and Steinberg (133) reported the isolation of mutants with an increased ability to take up 4-hydroxyphenylpyruvate, the mutations have not been mapped, and which transport systems are involved has not been established.
Dehydroshikimate and Shikimate.
The isolation of mutants showing either an increased (43, 95, 100) or decreased (262) capacity to transport shikimate may indicate the existence of a specific transport system. The ability of excess dehydroshikimate to inhibit shikimate uptake suggests that both compounds may be transported by the same system. The mutations lie near the his cluster of genes in a region referred to as shiA (43, 96). The number of specific genes and proteins involved in this proposed transport system has not been elaborated.
The role of the tnaB system seems fairly clear and different from the roles of the other four systems. TnaB synthesis is induced by tryptophan when cells are growing in conditions of no catabolite repression. Normally, the enzyme tryptophanase is induced at the same time, and this enzyme degrades the tryptophan that is transported into the cell. The capacity of the TnaB transporter to transport tryptophan is approximately 40 times greater than that of the Mtr system when both genes are expressed from the same promoter and with the same translation initiation region (J. Sarsero, unpublished data). This high capacity is clearly required to maintain internal pools of tryptophan in the presence of high levels of tryptophanase (350). The other four systems are primarily designed to provide aromatic amino acids for protein synthesis and perhaps to prevent the leakage of endogenous pools outside the cell. Regulation of the general transport system AroP is such that cells that are growing in minimal medium, i.e., in the absence of aromatic amino acids, are making this protein at an almost maximal rate (Table 4). Any aromatic amino acid in the environment can be transported by the system, and subsequent increases in intracellular pools will modulate expression of aroP, tyrP, and mtr. Because each of the aromatic amino acids can repress aroP expression and because each of the aromatic amino acids competes with the others to be carried by this transporter, the cell needs additional systems. High levels of a single aromatic amino acid can prevent the transport of the other two by AroP if they are present at lower levels. The synthesis of two of the specific systems, TyrP and Mtr, is also regulated by concentrations of amino acids inside the cell. In the presence of high intracellular levels of their individual substrates, tyrosine or tryptophan, both systems are shut down. However, they are derepressed if the intracellular concentrations of these amino acids fall, and their expression is further activated if, in the absence of tyrosine or tryptophan, the phenylalanine concentration is high. The activation of Mtr can be triggered by a high level of either phenylalanine or tyrosine. The interconnections between these systems may well have allowed maintenance of some constant relationship between the intracellular pools of each of these amino acids. One might speculate that failure to do so could result in unacceptable levels of misincorporation in protein synthesis.
I thank all my colleagues at Melbourne for permission to quote unpublished data and Helen Camakaris for critically reading the manuscript. I thank Charley Yanofsky for reading the section on TrpR-mediated control. Errors and omissions, however, remain my responsibility. I also thank Nadia Puglielli for typing the manuscript.
Work in my own laboratory has been supported by the Australian Research Council.
References
1. Adachi, O., L. D. Kohn, and E. W. Miles. 1974. Crystalline α2β2 complex of tryptophan synthetase of Escherichia coli. J. Biol. Chem. 249:7756–7763.
2. Adachi, O., and E. W. Miles. 1974. A rapid method for preparing crystalline β2 subunit of tryptophan synthetase of Escherichia coli in high yield. J. Biol. Chem. 249:5430–5434.
3. Adlersberg, M., and D. B. Sprinson. 1964. Syntheses of 3,7-dideoxy-d-threo-hepto-2,6-diulosonic acid: a study in 5-dehydroquinic acid formation. Biochemistry 3:1855–1860.
4. Ahmed, S. A., E. W. Miles, and D. R. Davies. 1985. Crystallization and preliminary X-ray, crystallographic data of the tryptophan synthase α2β2 complex from Salmonella typhimurium. J. Biol. Chem. 260:3716–3718.
5. Ahmed, S. A., S. B. Ruvinov, A. M. Kayastha, and E. W. Miles. 1991. Mechanism of mutual activation of the tryptophan synthase α and β subunits. J. Biol. Chem. 266:21548–21557.
6. Aksoy, S., C. L. Squires, and C. Squires. 1984. Translational coupling of the trpB and trpA genes in the Escherichia coli tryptophan operon. J. Bacteriol. 157:363–367.
7. Ames, G. F. 1964. Uptake of amino acids by Salmonella typhimurium. Arch. Biochem. Biophys. 104:1–18.
8. Ames, G. F., and J. R. Roth. 1968. Histidine and aromatic permeases of Salmonella typhimurium. J. Bacteriol. 96:1742–1749.
9. Andrews, A. E., B. Dickson, B. Lawley, C. Cobbett, and A. J. Pittard. 1991. Importance of position of TyrR boxes for repression and activation of the tyrP and aroF genes in Escherichia coli. J. Bacteriol. 173:5079–5085.
10. Andrews, A. E., B. Lawley, and A. J. Pittard. 1991. Mutational analysis of repression and activation of the tyrP gene in Escherichia coli. J. Bacteriol. 173:5068–5078.
11. Anton, J. A., and J. R. Coggins. 1988. Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase. Biochem. J. 249:319–326.
12. Argaet, V. P., T. J. Wilson, and B. E. Davidson. 1994. Purification of the Escherichia coli regulatory protein TyrR and analysis of its interaction with ATP, tyrosine, phenylalanine and tryptophan. J. Biol. Chem. 269:5171–5178.
13. Austin, S., and R. Dixon. 1992. The prokaryotic enhancer binding protein NtrC has an ATPase activity which is phosphorylation and DNA dependent. EMBO J. 11:2219–2228.
14. Austin, S., C. Kundrot, and R. Dixon. 1991. Influence of a mutation in the putative nucleotide binding site of the nitrogen regulatory protein NtrC on its positive control function. Nucleic Acids Res. 19:2281–2287.
15. Baasov, J., and J. R. Knowles. 1989. Is the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (tyrosine sensitive) a copper metallo-enzyme? J. Bacteriol. 171:6155–6160.
16. Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12, edition 7. Microbiol. Rev. 47:180–230.
17. Baker, T. I., and I. P. Crawford. 1966. Anthranilate synthetase. Partial purification and some kinetic studies on the enzyme from Escherichia coli. J. Biol. Chem. 241:5577–5584.
18. Balbinder, E. 1964. Intergeneric complementation between A and B components of bacterial tryptophan synthetase. Biochem. Biophys. Res. Commun. 17:770–774.
19. Baldwin, G. S., and B. E. Davidson. 1981. A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K-12 defective in the pheA gene. Arch. Biochem. Biophys. 211:66–75.
20. Baldwin, G. S., and B. E. Davidson. 1983. Kinetic studies on the mechanism of chorismate mutase/prephenate dehydratase from Escherichia coli K-12. Biochim. Biophys. Acta 742:374–383.
21. Baldwin, G. S., G. H. McKenzie, and B. E. Davidson. 1981. The self-association of chorismate mutase/prephenate dehydratase from Escherichia coli K-12. Arch. Biochem. Biophys. 211:76–85.
22. Ballou, C. E., H. O. L. Fischer, and D. L. MacDonald. 1955. The synthesis and properties of d-erythrose-4-phosphate. J. Am. Chem. Soc. 77:5967–5970.
23. Barker, D. G. 1982. Cloning and amplified expression of the tyrosyl-t-RNA synthetase genes of Bacillus stearothermophilis and Escherichia coli. Eur. J. Biochem. 125:357–360.
24. Barker, D. G., C. J. Bruton, and G. Winter. 1982. The tyrosyl-t-RNA synthetase from Escherichia coli. Complete nucleotide sequence of the structural gene. FEBS Lett. 150:419–423.
25. Bartholmes, P., H. Balk, and K. Kirschner. 1980. Mechanism of reconstitution of the apoβ2 subunit and the α2apoβ2 complex of tryptophan synthase with pyridoxal 5'-phosphate: kinetic studies. Biochemistry 19:4527–4533.
26. Bartholmes, P., K. Kirschner, and H. P. Gschwind. 1976. Cooperative and noncooperative binding of pyridoxal 5'-phosphate to tryptophan synthase from Escherichia coli. Biochemistry 15:4712–4717.
27. Bass, S., V. Sorrells, and P. Youderian. 1988. Mutant Trp repressors with new DNA-binding specificities. Science 242:240–245.
28. Bass, S., P. Sugiono, D. N. Arvidson, R. P. Gunsalus, and P. Youderian. 1987. DNA specificity determinants of Escherichia coli tryptophan repressor binding. Genes Dev. 1:565–572.
29. Bassegio, N., W. D. Davies, and B. E. Davidson. 1990. Identification of the promoter, operator and 5' and 3' ends of the mRNA of the Escherichia coli K-12 gene aroG. J. Bacteriol. 172:2547–2557.
30. Bauerle, R. H., and P. Margolin. 1966. A multifunctional enzyme complex in the tryptophan pathway of Salmonella typhimurium. Cold Spring Harbor Symp. Quant. Biol. 31:203–215.
31. Bedouelle, H., V. Guez, A. Vidal-Cros, and M. Herrmann. 1990. Overproduction of tyrosyl t-RNA synthetase is toxic to Escherichia coli: a genetic analysis. J. Bacteriol. 172:3940–3945.
32. Bennett, S. N., and C. Yanofsky. 1978. Sequence analysis of operator constitutive mutants of the tryptophan operon of Escherichia coli. J. Mol. Biol. 121:179–192.
33. Berlyn, M. D., and N. H. Giles. 1969. Organization of enzymes in the polyaromatic synthetic pathway: separability in bacteria. J. Bacteriol. 99:222–230.
34. Bisswanger, H., K. Kirschner, W. Cohn, V. Hager, and E. Hanssom. 1979. N-(5-Phosphoribosyl) isomerase-indoleglycerol-phosphate synthase. 1. A substrate analogue binds to two different binding sites on the bifunctional enzyme Escherichia coli. Biochemistry 18:5946–5953.
35. Blume, A., and E. Balbinder. 1966. The tryptophan operon of Salmonella typhimurium. Fine structure analysis by deletion mapping and abortive transduction. Genetics 53:577–592.
36. Bogosian, G., and R. Somerville. 1983. Trp repressor protein is capable of intruding into other amino acid biosynthetic systems. Mol. Gen. Genet. 191:51–58.
37. Bondinell, W. E., J. Vnek, P. F. Knowles, M. Sprecher, and D. B. Sprinson. 1971. On the mechanism of 5-enolpyruvylshikimate 3-phosphate synthetase J. Biol. Chem. 246:6191–6196.
38. Borg-Olivier, S. A., D. Tarlinton, and K. D. Brown. 1987. Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl tRNA synthetase operons. J. Bacteriol. 169:1949–1953.
39. Brennan, R. S., and B. W. Matthews. 1989. The helix-turn-helix DNA binding motif. J. Biol. Chem. 264:1903–1906.
40. Brown, K. D. 1968. Regulation of aromatic amino acid biosynthesis in Escherichia coli K-12. Genetics 60:31–48.
41. Brown, K. D. 1970. Formation of aromatic amino acid pools in Escherichia coli K-12. J. Bacteriol. 104:177–188.
42. Brown, K. D., and C. H. Doy. 1966. Control of three isoenzymic 7-phospho-2-oxo-3-deoxy-d-arabino-heptonate-d-erythrose-4-phosphate lyases of Escherichia coli W and derived mutants by repressive and "inductive" effects of the aromatic amino acids. Biochim. Biophys. Acta 118:157–172.
43. Brown, K. D., and C. H. Doy. 1976. Transport and utilization of the biosynthetic intermediate shikimic acid in Escherichia coli. Biochim. Biophys. Acta 428:550–562.
44. Brown, K. D., and R. L. Somerville. 1971. Repression of aromatic amino acid biosynthesis in Escherichia coli K-12. J. Bacteriol. 108:386–399.
45. Brzovic, P. J., Y. Saiva, C. C. Hyde, E. W. Miles, and M. F. Dunn. 1992. Evidence that mutations in a loop region of the α subunit inhibit the transition from an open to closed conformation in the tryptophan synthase bienzyme complex. J. Biol. Chem. 267:13028–13038.
46. Butler, J. R., W. L. Alworth, and M. J. Nugent. 1974. Mechanism of dehydroquinase catalyzed dehydration. J. Am. Chem. Soc. 96:1617–1618.
47. Caillet, J. 1990. Genetic mapping of pheV, an Escherichia coli gene for t-RNAPhe. Mol. Gen. Genet. 220:317–319.
48. Caillet, J., J. A. Plumbridge, and M. Springer. 1985. Evidence that pheV, a gene for tRNAPhe of E. coli, is transcribed from tandem promoters. Nucleic Acids Res. 13:3699–3710.
49. Caillet, J., J. A. Plumbridge, M. Springer, J. Vacher, C. Delamarche, R. N. Buckingham, and M. Grunberg-Manago. 1983. Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants. Nucleic Acids Res. 11:727–736.
50. Calender, R., and P. Berg. 1966. Purification and physical characterization of tyrosyl ribonucleic acid synthetases from Escherichia coli and Bacillus subtilis. Biochemistry 5:1681–1686.
51. Caligiuri, M. G., and R. Bauerle. 1991. The identification of amino acid residues involved in feedback regulation of the anthranilate synthase complex from Salmonella typhimurium. J. Biol. Chem. 266:8328–8335.
52. Caligiuri, M. G., and R. Bauerle. 1991. Subunit communication in the anthranilate synthase complex from Salmonella typhimurium. Science 252:1845–1848.
53. Camakaris, H., J. Camakaris, and J. Pittard. 1980. Regulation of aromatic amino acid biosynthesis in Escherichia coli K-12: control of the aroF-tyrA operon in the absence of repression control. J. Bacteriol. 143:613–620.
54. Camakaris, H., and J. Pittard. 1973. Regulation of tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: properties of the tyrR gene product. J. Bacteriol. 115:1135–1144.
55. Camakaris, H., and J. Pittard. 1982. Autoregulation of the tyrR gene. J. Bacteriol. 150:70–75.
56. Camakaris, J., and J. Pittard. 1971. Repression of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthetase (trp) and enzymes of the tryptophan pathway in Escherichia coli K-12. J. Bacteriol. 107:406–414.
57. Camakaris, J., and J. Pittard. 1974. Purification and properties of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthetase (trp) from Escherichia coli. J. Bacteriol. 120:590–597.
58. Carey, J., D. Lewis, T. Lavoie, and J. Yang. 1991. How does trp repressor recognize its operator. J. Biol. Chem. 266:24509–24513.
59. Charles, I. G., H. K. Lamb, D. Pickard, G. Dougan, and A. R. Hawkins. 1990. Isolation, characterisation and nucleotide sequences of the aroC genes encoding chorismate synthase from Salmonella typhi and Escherichia coli. J. Gen. Microbiol. 136:353–358.
60. Chaudhuri, S., and J. R. Coggins. 1985. The purification of shikimate dehydrogenase from Escherichia coli. Biochem. J. 226:217–223.
61. Chaudhuri, S., J. M. Lambert, L. A. McColl, and J. R. Coggins. 1986. Purification and characterisation of 3-dehydroquinase from Escherichia coli. Biochem. J. 239:699–704.
62. Chesne, S., A. Montmitonnet, and J. Pelmont. 1975. Transamination du l-aspartate et de la l-phenylalanine chez Escherichia coli K-12. Biochimie 57:1029–1034.
63. Chousterman, S., and G. Chaperville. 1973. Tyrosyl tRNA synthetase of Escherichia coli B. Eur. J. Biochem. 35:51–56.
64. Christie, G. E., and T. Platt. 1980. Gene structure in the tryptophan operon of Escherichia coli. Nucleotide sequence of trpC and the intercistronic regions. J. Mol. Biol. 142:519–530.
65. Chye, M.-L., J. R. Guest, and J. Pittard. 1986. Cloning of the aroP gene and identification of its product in Escherichia coli K-12. J. Bacteriol. 167:749–753.
66. Chye, M.-L., and J. Pittard. 1987. Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants. J. Bacteriol. 169:386–393.
67. Cobbett, C. S. 1983. Repression of the aroF promoter by the TyrR repressor Escherichia coli K-12: role of the "upstream" operator site. Mol. Microbiol. 2:377–383.
68. Cobbett, C. S., and M. T. Delbridge. 1987. Regulatory mutants of the aroF tyrA operon of Escherichia coli K-12. J. Bacteriol. 169:2500–2506.
69. Cobbett, C. S., and J. Pittard. 1980. Formation of a λ(Tn10) tyrR + specialized transducing bacteriophage from Escherichia coli K-12. J. Bacteriol. 144:877–883.
70. Cohen, G. 1983. The common pathway to lysine, methionine and threonine, p. 147–172. In K. M. Herrmann and R. L. Somerville (ed.), Amino Acids: Biosynthesis and Genetic Regulation. Addison-Wesley Publishing, Inc., Reading, Mass.
71. Cohen, G., and F. Jacob. 1959. Sur la repression de la synthése des enzymes intervenant dans la formation du tryptophane chez Escherichia coli. C.R. Acad. Sci. 248:3490–3492.
72. Cohn, W., K. Kirschner, and P. Paul. 1979. N-(5-Phosphoribosyl) anthranilate isomerase-indoleglycerol-phosphate synthase. 2. Fast reaction studies show that a fluorescent substrate analogue binds independently to two different sites. Biochemistry 18:5953–5959.
73. Collier, R. H., and G. Kohlhaw. 1972. Nonidentity of the aspartate and the aromatic aminotransferase components of transaminase A in Escherichia coli. J. Bacteriol. 112:365–371.
74. Comer, M. M., and A. Böck. 1976. Genes for the α and β subunits of the phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli. J. Bacteriol. 127:923–933.
75. Cornish, E. C., V. P. Argyropoulos, J. Pittard, and B. E. Davidson. 1986. Structure of the Escherichia coli K-12 regulatory gene tyrR: nucleotide sequence and sites of initiation and translation. J. Biol. Chem. 261:403–410.
76. Cornish, E. C., B. E. Davidson, and J. Pittard. 1982. Cloning and characterization of Escherichia coli K-12 regulator gene tyrR. J. Bacteriol. 152:1276–1279.
77. Cotton, R. G. H., and F. Gibson. 1965. The biosynthesis of phenylalanine and tyrosine: enzymes converting chorismic acid into prephenic acid and their relationships to prephenate dehydratase and prephenate dehydrogenase. Biochim. Biophys. Acta 100:76–88.
78. Crawford, I. P. 1975. Gene rearrangements in the evolution of the tryptophan synthetic pathway. Bacteriol. Rev. 39:87–120.
79. Crawford, I. P., B. P. Nichols, and C. Yanofsky. 1980. Nucleotide sequence of the trpB gene in Escherichia coli and Salmonella typhimurium. J. Mol. Biol. 142:489–502.
80. Crawford, I. P., and C. Yanofsky. 1958. On the separation of the tryptophan synthetase of Escherichia coli into two protein components. Proc. Natl. Acad. Sci. USA 44:1161–1170.
81. Creighton, T. E. 1970. N-(5'-Phosphoribosyl) anthranilate isomerase-indolyl-3-glycerol phosphate synthetase of tryptophan biosynthesis. Biochem. J. 120:699–707.
82. Creighton, T. E. 1970. A steady-state kinetic investigation of the reaction mechanism of the tryptophan synthetase of Escherichia coli. Eur. J. Biochem. 13:1–10.
83. Creighton, T. E. 1974. The functional significance of the evolutionary divergence between the tryptophan operons of Escherichia coli and Salmonella typhimurium. J. Mol. Evol. 4:121–137.
84. Creighton, T. E., D. R. Helinski, R. L. Somerville, and C. Yanofsky. 1966. Comparison of the tryptophan synthetase α subunits of several species of Enterobacteriaceae. J. Bacteriol. 91:1819–1826.
85. Creighton, T. E., and C. Yanofsky. 1966. Association of the α and β2 subunits of the tryptophan synthetase of Escherichia coli. J. Biol. Chem. 241:980–990.
86. Creighton, T. E., and C. Yanofsky. 1966. Indole-3-glycerol phosphate synthetase of Escherichia coli, an enzyme of the tryptophan operon J. Biol. Chem. 241:4616– 4624.
87. Cui, J., L. Ni, and R. L. Somerville. 1993. ATPase activity of TyrR, a transcriptional regulatory protein for σ70 RNA polymerase. J. Biol. Chem. 268:13023–13025.
88. Cui, J., and R. L. Somerville. 1993. Mutational uncoupling of the transcriptional activation function of the TyrR protein of Escherichia coli K-12 from the repression function. J. Bacteriol. 175:303–306.
89. Cui, J., and R. L. Somerville. 1993. The TyrR protein of Escherichia coli: analysis by limited proteolysis of domain structure and ligand-mediated conformational changes. J. Biol. Chem. 268:5040–5042.
90. Cui, J., and R. L. Somerville. 1993. A mutational analysis of the structural basis for transcriptional activation and monomer-monomer interaction in the TyrR system of Escherichia coli K-12. J. Bacteriol. 175:1777–1784.
91. Czernik, P. J., D. S. Shin, and B. K. Hurlburt. 1994. Functional selection and characterization of DNA binding sites for trp repressor of Escherichia coli. J. Biol. Chem. 269:27869–27875.
92. Dansette, P., and R. Azerad. 1974. The shikimate pathway. II. Stereospecificity of hydrogen transfer catalyzed by NADPH-dehydroshikimate reductase of E. coli. Biochimie 56:751–755.
93. Davidson, B. E., E. H. Blackburn, and T. A. A. Dopheide. 1972. Chorismate mutase-prephenate dehydratase from Escherichia coli K-12. Purification, molecular weight, and amino acid composition. J. Biol. Chem. 247:4441–4446.
94. Davies, W. D., and B. E. Davidson. 1982. The nucleotide sequence of aroG, the gene for 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase (phe) in Escherichia coli K-12. Nucleic Acids Res. 10:4045–4058.
95. Davis, B. D. 1950. Aromatic biosynthesis. I. The role of shikimic acid. J. Biol. Chem. 191:315–325.
96. Davis, B. D. 1952. Aromatic biosynthesis: antagonism between shikimic acid and its precursor 5-dehydroshikimic acid. J. Bacteriol. 64:749–757.
97. Davis, B. D. 1952. Aromatic biosynthesis. IV. Preferential conversion, in incompletely blocked mutants, of a common precursor of several metabolites. J. Bacteriol. 64:729–748.
98. Davis, B. D. 1953. Autocatalytic growth of a mutant due to accumulation of an unstable phenylalanine precursor. Science 118:251–252.
99. Davis, B. D., and E. S. Mingioli. 1953. Aromatic biosynthesis. VII. Accumulation of two derivatives of shikimic acid by bacterial mutants J. Bacteriol. 66:129–136.
100. Davis, B. D., and U. Weiss. 1953. Aromatic biosynthesis. VIII. The roles of 5-dehydroquinic acid and quinic acid. Arch. Exp. Pathol. Pharmacol. 220(Suppl.):1–15.
101. Dayan, J., and D. B. Sprinson. 1971. Enzyme alterations in tyrosine and phenylalanine auxotrophs of Salmonella typhimurium. J. Bacteriol. 108:1174–1180.
102. Deeb, G., K. Okamoto, and B. D. Hall. 1967. Isolation and characterization of nondefective transducing elements of bacteriophage φ80. Virology 31:288–295.
103. DeFeyter, R. C., B. E. Davidson, and J. Pittard. 1986. Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12. J. Bacteriol. 165:233–239.
104. DeFeyter, R. C., and J. Pittard. 1986. Genetic and molecular analysis of aroL, the gene for shikimate kinase II in Escherichia coli K-12. J. Bacteriol. 165:226–232.
105. DeFeyter, R. C., and J. Pittard. 1986. Purification and properties of shikimate kinase II from Escherichia coli K-12. J. Bacteriol. 165:331–333.
106. DeLeo, A. B., J. Dayan, and D. B. Sprinson. 1973. Purification and kinetics of tyrosine-sensitive 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthetase from Salmonella. J. Biol. Chem. 248:2344–2353.
107. DeLeo, A. B., and D. B. Sprinson. 1975. 3-Deoxy-d-arabino-heptulosonic acid 7-phosphate synthase mutants of Salmonella typhimurium. J. Bacteriol. 124:1312–1320.
108. Dopheide, T. A. A., P. Crewther, and B. E. Davidson. 1972. Chorismate mutase-prephenate dehydratase from Escherichia coli K-12. J. Biol. Chem. 247:4447–4452.
109. Doy, C. H. 1967. Tryptophan as an inhibitor of 3-deoxy-arabino-heptulosonate 7-phosphate synthetase. Biochem. Biophys. Res. Commun. 26:187–192.
110. Doy, C. H., and K. D. Brown. 1965. Control of aromatic biosynthesis: the multiplicity of 7-phospho-2-oxo-3-deoxy-d-arabino-heptonate d-erythrose-4-phosphate-lyase (pyruvate phosphorylating) in Escherichia coli W. Biochim. Biophys. Acta 104:377–389.
111. Doy, C. H., and F. Gibson. 1959. 1-(O-Carboxyphenylamino)-1-deoxyribulose. A compound formed by mutant strains of Aerobacter aerogenes and Escherichia coli blocked in the biosynthesis of tryptophan. Biochem. J. 72:586–597.
112. Doy, C., A. Rivera, and P. R. Srinivasan. 1961. Evidence for the enzymatic synthesis of N-(5'-phosphoribosyl) anthranilic acid, a new intermediate in tryptophan biosynthesis. Biochem. Biophys. Res. Commun. 4:83–88.
113. Duggleby, R. G., M. K. Snedden, and J. F. Morrison. 1978. Chorismate mutase-prephenate dehydratase from Escherichia coli: active sites of a bifunctional enzyme. Biochemistry 17:1548–1554.
114. Duncan, K., and J. R. Coggins. 1986. The serC-aroA operon of Escherichia coli. Biochem. J. 234:49–57.
115. Duncan, K., S. Chaudhuri, M. S. Campbell, and J. R. Coggins. 1986. The over expression and complete amino acid sequence of Escherichia coli 3-dehydroquinase. Biochem. J. 238:475–483.
116. Duncan, K., A. Lewendon, and J. R. Coggins. 1984. The complete amino acid sequence of Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase. FEBS Lett. 170:59–63.
117. Duncan, K., A. Lewendon, and J. R. Coggins. 1984. The purification of 5-enolpyruvylshikimate-3-phosphate synthase from an overproducing strain of Escherichia coli. FEBS Lett. 165:121–127.
118. Duncan, K., A. Lewendon, and J. R. Coggins. 1986. Evidence for a mixed operon encoding enzymes for two different pathways. Biochem. Soc. Trans. 14:263–264.
119. Dunn, M. F., V. Aguilar, P. Brzovic, W. F. Drewe, K. F. Houben, C. A. Leja, and M. Roy. 1990. The tryptophan synthase bienzyme complex transfers indole between the α and β sites via a 25–30A° long tunnel. Biochemistry 29:8598–8607.
120. Edwards, R. M., and M. D. Yudkin. 1982. Location of the gene for the low-affinity tryptophan-specific permease of Escherichia coli. Biochem. J. 204:617–619.
121. Egan, A. F., and F. Gibson. 1966. Partial purification of anthranilate synthase aggregate from A. aerogenes. Biochim. Biophys. Acta 130:276–277.
122. Ely, B., and J. Pittard. 1979. Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12. J. Bacteriol. 138:933–943.
123. Fayat, G., S. Blanquet, P. Dessin, G. Batches, and J. P. Waller. 1974. The molecular weight and subunit composition of phenylalanyl t-RNA synthetase from Escherichia coli K-12. Biochimie 56:35–41.
124. Fayat, G., J. F. Mayaux, C. Sacerdot, M. Fromant, M. Springer, M. Grunberg-Manago, and S. Blanquet. 1983. Escherichia coli phenylalanyl t-RNA synthetase operon region. Evidence for an attenuation mechanism. Identification of the gene for the ribosomal protein L20. J. Mol. Biol. 171:239–261.
125. Fotheringham, J. G., S. A. Dacey, P. P. Taylor, T. J. Smith, M. G. Hunter, M. E. Finlay, S. B. Primrose, D. M. Parker, and R. M. Edwards. 1986. The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K-12. Biochem. J. 234:593–604.
126. Frost, J. W., J. L. Binder, J. T. Kadonaga, and J. R. Knowles. 1984. Dehydroquinate synthase from Escherichia coli: purification, cloning and construction of overproducers of the enzyme. Biochemistry 23:4470–4475.
127. Gallagher, P. J., J. Schwartz, and D. Elseviers. 1984. Genetic mapping of pheU, an Escherichia coli gene for phenylalanyl tRNA. J. Bacteriol. 158:762–763.
128. Garner, C. C., and K. M. Herrmann. 1985. Operator mutations of the Escherichia coli aroF gene. J. Biol. Chem. 260:3820–3825.
129. Gavini, N., and B. E. Davidson. 1990. pheAo mutants of Escherichia coli have a defective pheA attenuator. J. Biol. Chem. 265:21532–21535.
130. Gavini, N., and B. E. Davidson. 1990. The pheR gene of Escherichia coli encodes t-RNAPhe, not a repressor protein. J. Biol. Chem. 265:21527–21531.
131. Gavini, N., and B. E. Davidson. 1991. Regulation of the pheA expression by the pheR product in Escherichia coli is mediated through attenuation of transcription. J. Biol. Chem. 266:7750–7753.
132. Gelfand, D. H., and N. Rudo. 1977. Mapping of the aspartate and aromatic amino acid aminotransferase genes tyrB and aspC. J. Bacteriol. 130:441–444.
133. Gelfand, D. H., and R. A. Steinberg. 1977. Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases J. Bacteriol. 130:429–440.
134. Gething, M.-J. H., and B. E. Davidson. 1976. Chorismate mutase/prephenate dehydratase from Escherichia coli. 2. Evidence for identical subunits catalysing the two activities. Eur. J. Biochem. 71:327–336.
135. Gething, M.-J. H., and B. E. Davidson. 1977. Chorismate mutase/prephenate dehydratase from Escherichia coli K-12. Eur. J. Biochem. 78:103–110.
136. Gething, M.-J. H., and B. E. Davidson. 1977. Chorismate mutase/prephenate dehydratase from Escherichia coli K-12. Effects of chemical modification on the enzymic activities and allosteric inhibition. Eur. J. Biochem. 78:111–117.
137. Gibson, F. 1964. Chorismic acid: purification and some chemical and physical studies. Biochem. J. 90:256–261.
138. Gibson, F., M. J. Jones, and H. Teltscher. 1955. Synthesis of indole and anthranilic acid by mutants of Escherichia coli. Nature (London) 175:853–854.
139. Gibson, F., and C. Yanofsky. 1960. The partial purification and properties of indole-3-glycerol phosphate synthetase from Escherichia coli. Biochim. Biophys. Acta 43:489–500.
140. Gibson, M. I., and F. Gibson. 1964. Preliminary studies on the isolation and metabolism of an intermediate in aromatic biosynthesis: chorismic acid. Biochem. J. 90:248–256.
141. Gicquel-Sanzey, B., and P. Cossart. 1982. Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action. EMBO J. 1:591–595.
142. Goldberg, M. E., T. E. Creighton, R. L. Baldwin, and C. Yanofsky. 1966. Subunit structure of tryptophan synthetase of Escherichia coli. J. Mol. Biol. 21:71–82.
143. Gollub, E. G., K. P. Liu, and D. B. Sprinson. 1973. tyrR, a regulatory gene of tyrosine biosynthesis in Salmonella typhimurium. J. Bacteriol. 115:1094–1102.
144. Gollub, E. G., K. P. Liu, and D. B. Sprinson. 1973. A regulatory gene of phenylalanine biosynthesis (pheR) in Salmonella typhimurium. J. Bacteriol. 115:121–128.
145. Gollub, E. G., and D. B. Sprinson. 1969. A regulatory mutation in tyrosine biosynthesis. Biochem. Biophys. Res. Commun. 35:389–395.
146. Gollub, E. G., H. Zalkin, and D. B. Sprinson. 1967. Correlation of genes and enzymes, and studies on regulation of the aromatic pathway in Salmonella. J. Biol. Chem. 242:5323–5328.
147. Gowrishankar, J., and J. Pittard. 1982. Construction from Mud1 (lac Ap r) lysogens of lambda bacteriophage bearing promoter-lac fusions: isolation of λpheA-lac. J. Bacteriol. 150:1122–1129.
148. Gowrishankar, J., and J. Pittard. 1982. Regulation of phenylalanine biosynthesis in Escherichia coli K-12: control of transcription of the pheA operon. J. Bacteriol. 150:1130–1137.
149. Gowrishankar, J., and J. Pittard. 1982. Molecular cloning of pheR in Escherichia coli K-12. J. Bacteriol. 152:1–6.
150. Grove, C. L., and R. P. Gunsalus. 1987. Regulation of the aroH operator by the tryptophan repressor. J. Bacteriol. 169:2158–2164.
151. Gschwind, H. P., V. Gschwind, C. H. Paul, and K. Kirschner. 1979. Affinity chromotography of tryptophan synthase from Escherichia coli: systematic studies with immobilized tryptophanol phosphate. Eur. J. Biochem. 96:403–416.
152. Gunsalus, R. P., and C. Yanofsky. 1980. Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. Proc. Natl. Acad. Sci. USA 77:7117–7121.
153. Gunsalus, R. P., S. Zurawski, and C. Yanofsky. 1979. Structural and functional analysis of cloned deoxyribonucleic acid containing the trpR-thr region of the Escherichia coli chromosome. J. Bacteriol. 140:106–113.
154. Hall, C. V., M. vanCleemput, K. H. Muench, and C. Yanofsky. 1982. The nucleotide sequence of the structural gene for Escherichia coli tryptophanyl t-RNA synthetase. J. Biol. Chem. 257:6132–6136.
155. Hall, C. V., and C. Yanofsky. 1982. Regulation of tryptophanyl t-RNA synthetase formation. J. Bacteriol. 151:918–923.
156. Hanson, K. R., and I. A. Rose. 1963. The absolute stereochemical course of citric acid biosynthesis. Proc. Natl. Acad. Sci. USA 50:981–988.
157. Haran, T. E., A. Joachimiak, and P. B. Sigler. 1992. The DNA target of the trp repressor. EMBO J. 11:3021–3030.
158. Hathaway, G. M., and I. P. Crawford. 1970. Studies on the association of β-chain monomers of Escherichia coli tryptophan synthetase. Biochemistry 9:1801–1808.
159. Hathaway, G. M., S. Kida, and I. P. Crawford. 1969. Subunit structure of the B component of Escherichia coli tryptophan synthetase. Biochemistry 8:989–996.
160. He, B., and H. Zalkin. 1992. Repression of Escherichia coli purB is by a transcriptional road block mechanism. J. Bacteriol. 174:7121–7127.
161. Heatwole, V. M., and R. L. Somerville. 1991. The tryptophan-specific permease gene, mtr, is differentially regulated by tryptophan and tyrosine repressor in Escherichia coli K-12. J. Bacteriol. 173:3601–3604.
162. Heatwole, V. M., and R. L., Somerville. 1991. Cloning, nucleotide sequence and characterisation of mtr, the structural gene for the tryptophan-specific permease of Escherichia coli K-12. J. Bacteriol. 173:108–115.
163. Heatwole, V. M., and R. L. Somerville. 1992. Synergism between the Trp repressor and Tyr repressor in repression of aroL promoter of Escherichia coli K-12. J. Bacteriol. 174:331–335.
164. Henderson, E. J., H. Nagano, H. Zalkin, and L. H. Hwang. 1970. The anthranilate synthetase-anthranilate 5-phosphoribosyl-pyrophosphate phosphoribosyl-transferase aggregate. J. Biol. Chem. 245:1416–1423.
165. Henderson, E. J., and H. Zalkin. 1971. On the composition of anthranilate synthetase-anthranilate 5-phosphoribosyl-pyrophosphate phosphoribosyl-transferase from Salmonella typhimurium. J. Biol. Chem. 246:6891–6898.
166. Henning, J., D. R. Helinski, F. B. Chao, and C. Yanofsky. 1960. Isolation and crystallization of A protein of tryptophan synthetase of Escherichia coli. J. Biol. Chem. 237:1523–1530.
167. Herrmann, K. M., J. Shultz, and M. A. Hermodson. 1980. Sequence homology between tyrosine sensitive 3-deoxy d-arabino heptulosonate 7-phosphate synthase from Escherichia coli and hemerythrin from Sipunculida. J. Biol. Chem. 255:7079–7081.
167a. Herrmann, K. M., and R. L. Somerville (ed.). 1983. Amino Acids: Biosynthesis and Genetic Regulation. Addison-Wesley Publishing Co., Reading, Mass.
168. Aerobacter aerogenes. Evidence that the two reactions occur at one active site. Biochemistry 18:2766–2775.
169. Heyde, E., and J. F. Morrison. 1978. Kinetic studies on the reactions catalyzed by chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes. Biochemistry 17:1573–1580.
170. Högberg-Raibaud, A., and M. E. Goldberg. 1977. Preparation and characterization of a modified form of β2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding. Proc. Natl. Acad. Sci. USA 74:442–446.
171. Hoiseth, S. K., and B. A. D. Stocker. 1985. Genes aroA and serC of Salmonella typhimurium constitute an operon. J. Bacteriol. 163:355–361.
172. Honore, N., and S. T. Cole. 1990. Nucleotide sequence of the aroP gene encoding the general aromatic amino acid transport protein of Escherichia coli K-12: homology with yeast transport proteins. Nucleic Acids Res. 18:653.
173. Hu, C.-Y., and D. B. Sprinson. 1977. Properties of tyrosine-inhibitable 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthase from Salmonella. J. Bacteriol. 129:177–183.
174. Hudson, G. S., and B. E. Davidson. 1984. Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K-12. J. Mol. Biol. 180:1023–1051.
175. Hudson, G. S., G. J. Howlett, and B. E. Davidson. 1983. The binding of tyrosine and NAD+ to chorismate mutase/prephenate dehydrogenase from Escherichia coli K-12 and the effects of these ligands on the activity and self-association of the enzyme. J. Biol. Chem. 258:3114–3120.
176. Hudson, G. S., P. Rellos, and B. E. Davidson. 1991. Two promoters control the aroH gene of Escherichia coli. Gene 102:87–89.
177. Hudson, G. S., V. Wong, and B. E. Davidson. 1985. Chorismate mutase-prephenate dehydrogenase from Escherichia coli K-12. Purification, characterisation and identification of a reactive cysteine. Biochemistry 23:6240–6249.
178. Hwang, L. H., and H. Zalkin. 1971. Multiple forms of anthranilate synthetase-anthranilate 5-phosphoribosyl-pyrophosphate phosphoribosyl-transferase from Salmonella typhimurium. J. Biol. Chem. 246:2338–2345.
179. Hyde, S. C., S. A. Ahmed, E. A. Pallars, E. H. Miles, and D. R. Davies. 1988. Three dimensional structure of the tryptophan synthase α2β2 multienzyme complex from Salmonella typhimurium. J. Biol. Chem. 263:17857–17871.
180. Im, S. W. K., H. Davidson, and J. Pittard. 1971. Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-d-arabinoheptulosonic acid 7-phosphate synthetase (Phe), 3-deoxy-d-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A. J. Bacteriol. 108:400–409.
181. Im, S. W. K., and J. Pittard. 1973. Tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: mutants derepressed for chorismate mutase P-prephenate dehydratase. J. Bacteriol. 106:784–790.
182. Im, S. W. K., and J. Pittard. 1973. Tyrosine and phenylalanine biosynthesis in Escherichia coli K-12: complementation between different tyrR alleles. J. Bacteriol. 115:1145–1150.
183. Ishihama, A. 1993. Protein-protein communication within the transcription apparatus. J. Bacteriol. 175:2483–2487.
184. Ito, J., E. C. Cox, and C. Yanofsky. 1969. Anthranilate synthetase, an enzyme specified by the tryptophan operon of Escherichia coli: purification and characterization of component I. J. Bacteriol. 97:725–733.
185. Ito, J., and I. P. Crawford. 1965. Regulation of the enzymes of the tryptophan pathway in Escherichia coli. Genetics 52:1303–1316.
186. Ito, J., and C. Yanofsky. 1966. The nature of the anthranilic acid synthetase complex of Escherichia coli. J. Biol. Chem. 241:4113–4114.
187. Jackson, E. N., and C. Yanofsky. 1973. The region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function. J. Mol. Biol. 76:89–101.
188. Jensen, R. A., D. S. Nasser, and E. W. Nester. 1967. Comparative control of a branch point enzyme in microorganisms. J. Bacteriol. 94:1582–1593.
189. Joachimiak, A., R. L. Kelley, R. P. Gunsalus, C. Yanofsky, and P. B. Sigler. 1983. Purification and characterization of trp aporepressor. Proc. Natl. Acad. Sci. USA 80:668–672.
190. Joseph, D. R., and K. H. Muench. 1971. Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. J. Biol. Chem. 246:7602–7609.
191. Kaback, H. R., K. Jung, H. Jung, J. Wu, G. G. Prive, and K. Zen. 1993. What’s new with lactose permease. J. Bioenerg. Biomembr. 25:627–636.
192. Kasian, P. A., B. E. Davidson, and J. Pittard. 1986. Molecular analysis of the promoter operator region of the Escherichia coli K-12 tyrP gene. J. Bacteriol. 167:556–561.
193. Katagiri, M., and R. Sato. 1953. Accumulation of phenylalanine by a phenylalanineless mutant of Escherichia coli. Science 118:250–251.
194. Kelley, R. L., and C. Yanofsky. 1985. Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA. Proc. Natl. Acad. Sci. USA 82:483–487.
195. Kinghorn, J. R., M. Schweizer, N. H. Giles, and S. R. Kushner. 1981. The cloning and analyses of the aroD gene of Escherichia coli K-12. Gene 14:73–80.
196. Kirschner, K., H. Szadkowski, A. Henschen, and F. Lottspeich. 1980. Limited proteolysis of N-(5'-phosphoribosyl) anthranilate isomerase:indole glycerol phosphate synthase from Escherichia coli yields two different enzymically active, functional domains. J. Mol. Biol. 143:395–409.
197. Kirschner, K., R. L. Wiskocil, M. Foehn, and L. Rizean. 1975. The tryptophan synthase from Escherichia coli. Eur. J. Biochem. 60:513–523.
198. Klig, L. S., J. Carey, and C. Yanofsky. 1988. trp repressor interactions with the trp aroH and trpR operators. J. Mol. Biol. 202:764–777.
199. Koch, G. L. E., D. C. Shaw, and F. Gibson. 1971. The purification and characterization of chorismate mutase-prephenate dehydrogenase from Escherichia coli K-12. Biochim. Biophys. Acta 229:795–804.
200. Koch, G. L. E., D. C. Shaw, and F. Gibson. 1971. Characterization of the subunits of chorismate mutase-prephenate dehydrogenase from Escherichia coli K-12. Biochim. Biophys. Acta 229:805–812.
201. Koch, G. L. E., D. C. Shaw, and F. Gibson. 1972. Studies on the relationship between the active sites of chorismate mutase-prephenate dehydrogenase from Escherichia coli or Aerobacter aerogenes. Biochim. Biophys. Acta 258:719–730.
202. Kohara, Y., K. Akiyama, and K. Isono. 1987. The physical map of the whole Escherichia coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50:495–508.
203. Kondo, K., S. Wakabayashi, T. Yagi, and H. Kagamiyama. 1984. The complete amino acid sequence of aspartate aminotransferase from Escherichia coli: sequence comparison with pig isoenzymes. Biochem. Biophys. Res. Commun. 122:62–67.
204. Kumamoto, A. A., W. S. Miller, and R. P. Gunsalus. 1987. Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators. Genes Dev. 1:556–564.
205. Kuramitsu, S., T. Ogawa, H. Ogawa, and H. Kagamiyama. 1985. Branched chain amino acid aminotransferase of Escherichia coli: nucleotide sequence of the ilvE gene and deduced amino acid sequence. J. Biochem. 97:993–999.
206. Kuramitsu, S., S. Okumo, T. Ogawa, H. Ogawa, and H. Kagamiyama. 1985. Aspartate aminotransferase of Escherichia coli: nucleotide sequence of the aspC gene. J. Biochem. 97:1259–1262.
207. Lam, H. M., and M. E. Winkler. 1992. Characterisation of the complex pdxH-tyrS operon of Escherichia coli K-12 and pleiotropic phenotypes caused by pdxH insertion mutations. J. Bacteriol. 174:6033–6045.
208. Lawley, B., and A. J. Pittard. 1994. Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12. J. Bacteriol. 176:6921–6930.
209. Lawson, C. L., and J. Carey. 1993. Tandem binding in crystals of a trp repressor/operator half-site complex. Nature (London) 366:178–182.
210. LeMarechal, P., and R. Azerad. 1976. The shikimate pathway. III. 3-Dehydroquinate synthetase of E. coli. Mechanistic studies by kinetic isotope effect. Biochimie 58:1123–1128.
211. LeMarechal, P., C. Froussios, M. Level, and R. Azerad. 1981. Synthesis of phosphono analogues of 3-deoxy-d-arabino-hept-2-ulosonic acid 7-phosphate. Carbohydr. Res. 94:1–10.
212. Levin, J. G., and D. B. Sprinson. 1960. The formation of 3-enolpyruvyl shikimate 5-phosphate in extracts of Escherichia coli. Biochem. Biophys. Res. Commun. 3:157–163.
213. Levin, J. G., and D. B. Sprinson. 1964. The enzymatic formation and isolation of 3-enolpyruvylshikimate 5-phosphate. J. Biol. Chem. 239:1142–1150.
214. Lewendon, A., and J. R. Coggins. 1983. Purification of 5-enolpyruvyl shikimate 3-phosphate synthase from Escherichia coli. Biochem. J. 213:187–191.
215. Li, S. L., and C. Yanofsky. 1972. Amino acid sequences of fifty residues from the amino termini of the tryptophan synthetase α chains of several enterobacteriaceae. J. Biol. Chem. 247:1031–1037.
216. Liu, Y. C., and K. S. Matthews. 1993. Dependence of trp repressor-operator affinity, stoichiometry and apparent cooperativity on DNA sequence and size. J. Biol. Chem. 268:23239–23249.
217. Löbner-Oleson, A. and M. G. Marinus. 1992. Identification of the gene (aroK) encoding shikimic acid kinase I of Escherichia coli. J. Bacteriol. 174:525–529.
218. Lowry, O. H., J. Carter, J. B. Ward, and L. Glaser. 1971. The effect of carbon and nitrogen sources on the level of metabolic intermediates in Escherichia coli. J. Biol. Chem. 246:6511–6521.
219. Luisi, B. F., and P. B. Sigler. 1990. The sterochemistry and biochemistry of the trp repressor-operator complex. Biochim. Biophys. Acta 1048:113–126.
220. Maitra, U. S., and D. B. Sprinson. 1978. 5-Dehydro-3-deoxy-d-arabino-heptulosonic acid 7-phosphate. An intermediate in the 3-dehydroquinate synthase reaction. J. Biol. Chem. 253:5426–5430.
221. Marmorstein, R. Q., and P. B. Sigler. 1989. Stereochemical effects of l-tryptophan and its analogues on trp repressor’s affinity for operator DNA. J. Biol. Chem. 269:9149–9154.
222. Matsushiro, A. 1963. Specialized transduction of tryptophan markers in Escherichia coli K-12: isolation and characterization of operator mutants. J. Bacteriol. 107:8–15.
223. Mattern, I. E., and J. Pittard. 1971. Regulation of tyrosine biosynthesis in Escherichia coli K-12: isolation and characterization of operator mutants. J. Bacteriol. 107:8–15.
224. Mavrides, C., and W. Orr. 1974. Multiple forms of plurispecific aromatic: 2-oxoglutarate (oxaloacetate) aminotransferase (transaminase A) in Escherichia coli and selective repression by l-tyrosine. Biochim. Biophys. Acta 336:70–78.
225. Mavrides, C., and W. Orr. 1975. Multispecific aspartate and aromatic amino acid aminotransferases in Escherichia coli. J. Biol. Chem. 250:4128–4133.
226. Mayaux, J. F., G. Fayat, M. Panvert, M. Springer, M. Grunberg-Manago, and S. Blanquet. 1985. Control of phenylalanyl-t-RNA synthethase genetic expression. Site-directed mutagenesis of the pheST operon regulatory region "in vitro." J. Mol. Biol. 184:31–44.
227. McCandliss, R. J., and K. M. Herrmann. 1978. Iron, an essential element for the biosynthesis of aromatic compounds. Proc. Natl. Acad. Sci. USA 75:4810–4813.
228. McCandliss, R. J., M. D. Poling, and K. M. Herrmann. 1978. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine sensitive isoenzyme from Escherichia coli. J. Biol. Chem. 253:4259–4265.
229. Mechulam, Y., G. Fayat, and S. Blanquet. 1985. Sequence of the Escherichia coli, pheST operon and identification of the himA gene. J. Bacteriol. 163:787–791.
230. Médigue, C., A. Viari, A. Hénant, and A. Danchin. 1993. Colibri: a functional data base for the Escherichia coli genome. Microbiol. Rev. 57:623–654.
231. Miles, E. W. 1979. Tryptophan synthase: structure, function and subunit interaction. Adv. Enzymol. 49:127–186.
232. Miles, E. W. 1980. Tryptophan synthase: structure, function and interaction with d-tryptophan and l-tryptophan, p. 137–147. In D. Hayaishi, Y. Ishimara, and R. Kido (ed.), Biochemical and Medical Aspects of Tryptophan Metabolism. Elsevier-North Holland, Amsterdam.
233. Miles, E. W., and M. Moriguchi. 1977. Tryptophan synthase of Escherichia coli. J. Biol. Chem. 252:6594–6599.
234. Millar, G., and J. R. Coggins. 1986. The complete amino acid sequence of 3-dehydroquinate synthase of Escherichia coli K-12. FEBS Lett. 200:11–17.
235. Millar, G., A. Lewendon, M. C. Hunter, and J. R. Coggins. 1986. The cloning and expression of the aroL gene from Escherichia coli. Biochem. J. 237:427–437.
236. Mitsuhashi, S., and B. D. Davis. 1954. Aromatic biosynthesis. XII. Conversion of 5-dehydroquinic acid to 5-dehydroshikimic acid by 5-dehydroquinase. Biochim. Biophys. Acta 15:54–61.
237. Moldovani, I. S., and G. Denes. 1968. Mechanism of the action and of the allosteric inhibition of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (tyrosine sensitive) of Escherichia coli W. Acta Biochim. Biophys. Acad. Sci. Hung. 3:259–273.
238. Monnier, N., A. Montmitonnet, S. Chesne, and J. Pelmont. 1976. Transaminase B d’ Escherichia coli. I. Purification et premières propriétés. Biochimie 58:663–675.
239. Morell, H., M. J. Clark, P. F. Knowles, and D. B. Sprinson. 1967. The enzymic synthesis of chorismic and prephenic acids from 3-enolpyruvyl shikimic acid 5-phosphate. J. Biol. Chem. 242:82–90.
240. Morell, H., and D. B. Sprinson. 1968. Shikimate kinase isoenzymes in Salmonella typhimurium. J. Biol. Chem. 243:676–677.
241. Mosteller, R. D., K. R. Nishimoto, and R. V. Goldstein. 1977. Inactivation and partial degradation of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase in non-growing cultures of Escherichia coli. J. Bacteriol. 131:153–162.
242. Muday, G. T., D. J. Johnson, R. L. Somerville, and K. M. Herrmann. 1991. The tyrosine repressor negatively regulates aroH expression in Escherichia coli. J. Bacteriol. 173:3930–3932.
243. Muench, V. H. 1976. Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. J. Biol. Chem. 251:5195–5199.
244. Murphy, T. M., and S. E. Mills. 1969. Immunochemical and enzymatic comparisons of the tryptophan synthase α subunits from five species of Enterobacteriaceae. J. Bacteriol. 97:1310–1320.
245. Nagano, H., and H. Zalkin. 1970. Some physicochemical properties of anthranilate synthetase component I from Salmonella typhimurium. J. Biol. Chem. 245:3097–3130.
246. Neidhardt, F. C., P. L. Bloch, S. Pedersen, and S. Rich. 1977. Chemical measurement of steady-state levels of ten amino acyl-transfer ribonucleic acid synthetases in Escherichia coli. J. Bacteriol. 129:378–387.
247. Nichols, B. P., G. F. Miozzari, M. van Cleemput, G. N. Bennett, and C. Yanofsky. 1980. Nucleotide sequences of the trpG regions of Escherichia coli, Shigella dysenteriae, Salmonella typhimurium and Serratia marcescens. J. Mol. Biol. 142:503–517.
248. Nichols, B. P., and C. Yanofsky. 1979. Nucleotide sequences of trpA of Salmonella typhimurium and Escherichia coli: an evolutionary comparison. Proc. Natl. Acad. Sci. USA 76:5244–5248.
249. Nishioka, Y., M. Demerec, and A. Eisenstark. 1967. Genetic analysis of aromatic mutants of Salmonella typhimurium. Genetics 56:341–351.
250. Ogino, T., C. Garner, J. L. Markley, and K. M. Herrmann. 1982. Biosynthesis of aromatic compounds: 13C NMR spectroscopy of whole Escherichia coli cells. Proc. Natl. Acad. Sci. USA 79:5828–5832.
251. Oppenheim, D. S., and C. Yanofsky. 1980. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics 95:785–795.
252. Otwinowski, Z., R. W. Schevitz, R. S. Zhang, C. L. Lawson, A. Joachimiak, R. Q. Marmorstein, B. F. Luisi, and P. B. Sigler. 1988. Crystal structure of trp repressor/operator complex at atomic resolution. Nature (London) 335:321–329.
253. Pabst, M. J., J. C. Kuhn, and R. L. Somerville. 1973. Feedback regulation in the anthranilate aggregate from wild type and mutant strains of Escherichia coli. J. Biol. Chem. 248:901–914.
254. Pfau, J., D. N. Arvidson, and P. Youderian. 1994. Mutants of Escherichia coli Trp repressor with changes of conserved, helix-turn-helix residue threonine 81 have altered DNA-binding specificities. Mol. Microbiol. 13:1001–1012.
255. Pi, J., P. J. Wookey, and A. J. Pittard. 1991. Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli. J. Bacteriol. 173:3622–3629.
256. Pi, J., P. J. Wookey, and A. J. Pittard. 1993. Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli. J. Bacteriol. 175:7500–7504.
257. Pittard, A. J., and B. E. Davidson. 1991. TyrR protein of Escherichia coli and its role as repressor and activator. Mol. Microbiol. 5:1585–1592.
258. Pittard, J., J. Camakaris, and B. J. Wallace. 1969. Inhibition of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthetase (Trp) in Escherichia coli. J. Bacteriol. 97:1242–1247.
259. Pittard, J., and F. Gibson. 1970. The regulation of aromatic amino acids and vitamins. Curr. Top. Cell. Regul. 2:29–63.
260. Pittard, J., J. Praszkier, A. Certoma, G. Eggertsson, J. Gowrishankar, G. Narasiah, and M. J. Whipp. 1990. Evidence that there are only two t-RNAPhe genes in Escherichia coli. J. Bacteriol. 172:6077–6083.
261. Pittard, J., and B. J. Wallace. 1966. Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli. J. Bacteriol. 91:1494–1508.
262. Pittard, J., and B. J. Wallace. 1966. Gene controlling the uptake of shikimic acid by Escherichia coli. J. Bacteriol. 92:1070–1075.
263. Pizer, L. I., and M. L. Potochny. 1964. Nutritional and regulatory aspects of serine metabolism in Escherichia coli. J. Bacteriol. 88:611–619.
264. Platt, T., and C. Yanofsky. 1975. An intercistronic region and ribosome binding site in bacterial messenger RNA. Proc. Natl. Acad. Sci. USA 72:2399–2403.
265. Plumbridge, J. A., and M. Springer. 1986. Genes for the two subunits of phenylalanyl t-RNA synthetase of Escherichia coli are transcribed from the same promoter. J. Mol. Biol. 144:595–600.
266. Powell, J. T., and J. F. Morrison. 1978. The purification and properties of the aspartate aminotransferase and aromatic amino acid aminotransferase from Escherichia coli. Eur. J. Biochem. 87:391–400.
267. Powell, J. T., and J. F. Morrison. 1979. Enzyme-enzyme interaction and the biosynthesis of the aromatic amino acids in Escherichia coli. Biochim. Biophys. Acta 568:467–474.
268. Ray, J. M. and R. Bauerle. 1991. Purification and properties of tryptophan sensitive 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase from Escherichia coli. J. Bacteriol. 173:1894–1901.
269. Ray, J. M., C. Yanofsky, and R. Bauerle. 1988. Mutational analysis of the catalytic and feedback sites of the tryptophan sensitive 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase of Escherichia coli. J. Bacteriol. 170:5500–5506.
270. Reizer, J., Finley, K., D. Kakuda, C. L. MacLeod, A. Reizer, and M. H. Saier, Jr. 1993. Mammalian integral membrane receptors are homologous to facilitators and antiporters of yeast, fungi and eubacteria. Protein Sci. 2:20–30.
271. Rivera, A., Jr., and P. R. Srinivasan. 1962. 3-Enolpyruvylshikimate 5-phosphate, an intermediate in the biosynthesis of anthranilate. Proc. Natl. Acad. Sci. USA 48:864–867.
272. Roeder, W., and R. L. Somerville. 1979. Cloning the trpR gene. Mol. Gen. Genet. 176:361–368.
273. Rood, J. I., B. Perrot, E. Heyde, and J. F. Morrison. 1982. Characterisation of monofunctional chorismate mutase/prephenate dehydrogenase enzymes obtained via mutagenesis of recombinant plasmids in vivo. Eur. J. Biochem. 124:513–519.
274. Rose, J. K., C. L. Squires, C. Yanofsky, H. L. Yang, and G. Zubay. 1973. Regulation of "in vitro" transcription of the tryptophan operon by purified RNA polymerase in the presence of partially purified repressor and tryptophan. Nature (London) New Biol. 245:133–137.
275. Rose, J. K., and C. Yanofsky. 1972. Metabolic regulation of the tryptophan operon of Escherichia coli: repressor-independent regulation of transcription initiation frequency. J. Mol. Biol. 69:103–118.
276. Rotenberg, S. L., and D. B. Sprinson. 1970. Mechanism and sterochemistry of 5-dehydroquinate synthetase. Proc. Natl. Acad. Sci. USA 67:1669–1672.
277. Rotenberg, S. L., and D. B. Sprinson. 1978. Isotope effects in 3-dehydroquinate synthase and dehydratase. J. Biol. Chem. 253:2210–2215.
278. Rudman, D., and A. Meister. 1953. Transamination in Escherichia coli. J. Biol. Chem. 200:591–604.
279. Salamon, I. I., and B. D. Davis. 1953. Aromatic biosynthesis. IX. The isolation of a precursor of shikimic acid. J. Am. Chem. Soc. 75:5567–5571.
280. Sampathkumar, P., and J. F. Morrison. 1982. Chorismate mutase-prephenate dehydrogenase from Escherichia coli. Purification and properties of the bifunctional enzyme. Biochim. Biophys. Acta 702:204–211.
281. Sampathkumar, P., and J. F. Morrison. 1982. Chorismate mutase-prephenate dehydrogenase from Escherichia coli. Kinetic mechanism of the prephenate dehydrogenase reaction. Biochim. Biophys. Acta 702:212–219.
282. Sanderson, K. E., and J. R. Roth. 1983. Linkage map of Salmonella typhimurium, edition VI. Microbiol. Rev. 47:410–453.
283. Sarsero, J. P., and A. J. Pittard. 1991. Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12. J. Bacteriol. 173:7701–7704.
284. Sarsero, J. P., and A. J. Pittard. 1995. Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and β-galactosidase fusions. J. Bacteriol. 177:297–306.
285. Sarsero, J. P., P. J. Wookey, P. Gollnick, C. Yanofsky, and A. J. Pittard. 1991. A new family of integral membrane proteins involved in the transport of aromatic amino acids in Escherichia coli. J. Bacteriol. 173:3231–3234.
286. Sarsero, J. P., P. J. Wookey, and A. J. Pittard. 1991. Regulation of expression of the Escherichia coli K-12 mtr gene by TyrR protein and Trp repressor. J. Bacteriol. 173:4133–4143.
287. Schevitz, R. W., Z. Otwinowski, A. Joachimiak, C. L. Lawson, and P. B. Sigler. 1985. The three dimensional structure of trp repressor. Nature (London) 317:782–786.
288. Schmit, J. C., S. W. Artz, and H. Zalkin. 1970. Chorismate mutase-prephenate dehydrogenase. J. Biol. Chem. 245:4019–4027.
289. Schmit, J. C., and H. Zalkin. 1969. Chorismate mutase-prephenate dehydratase. Partial purification and properties of the enzyme from Salmonella typhimurium. Biochemistry 8:174–181.
290. Schmit, J. C., and H. Zalkin. 1971. Chorismate mutase-prephenate dehydratase. Phenylalanine induced dimerization and its relationship to feedback inhibition. J. Biol. Chem. 246:6002–6010.
291. Schoner, R., and K. M. Herrmann. 1976. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase. J. Biol. Chem. 251:5440–5447.
292. Schultz, G. E., and T. E. Creighton. 1969. Preliminary X-ray diffraction study of the wild-type and mutationally altered tryptophan synthetase α subunit. Eur. J. Biochem. 10:195–197.
293. Schwartz, J., R. Klotsky, P. J. Gallagher, M. Krauskoff, M. A. Q. Siddiqui, J. F. H. Wong, and B. A. Roc. 1983. Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe. Nucleic Acids Res. 11:4379–4389.
294. Selker, E., and C. Yanofsky. 1979. Nucleotide sequence of the trpC-trpB intercistronic region from Salmonella typhimurium. J. Mol. Biol. 130:135–143.
295. Shultz, J., M. A. Hermodson, C. C. Garner, and K. M. Herrmann. 1984. The nucleotide sequence of the aroF gene of Escherichia coli and the amino acid sequence of the encoded protein, the tyrosine-sensitive 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase. J. Biol. Chem. 259:9655–9661.
296. Shultz, J., M. A. Hermodson, and K. M. Herrmann. 1981. A comparison of the amino-terminal sequences of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Escherichia coli. FEBS Lett. 131:108–110.
297. Silbert, D. F., S. E. Jorgensen, and E. C. C. Lin. 1963. Repression of transaminase A by tyrosine in Escherichia coli. Biochim. Biophys. Acta 73:232–240.
298. Simmonds, S. 1950. The metabolism of phenylalanine and tyrosine in mutant strains of Escherichia coli. J. Biol. Chem. 185:755–762.
299. Simpson, R. J., and B. E. Davidson. 1976. Studies on 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase (phe) from Escherichia coli K-12. Eur. J. Biochem. 70:501–507.
300. Smith, L. C., J. M. Ravel, S. R. Lax, and W. Shive. 1962. The control of 3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthesis by phenylalanine and tyrosine. J. Biol. Chem. 237:3566–3570.
301. Smith, O. H. 1967. Structure of the trpC cistron specifying indoleglycerol phosphate synthetase, and its localization in the tryptophan operon of Escherichia coli. Genetics 57:95–105.
302. Smith, O. H., and C. Yanofsky. 1960. 1-(O-Carboxyphenylamino)-1-deoxyribulose 5-phosphate. A new intermediate in the biosynthesis of tryptophan. J. Biol. Chem. 235:2051–2057.
303. Somerville, R. 1992. The Trp repressor, a ligand-activated regulatory protein. Prog. Nucleic Acids Res. Mol. Biol. 42:1–38.
304. Springer, M., J. F. Mayaux, G. Fayat, J. A. Plumbridge, M. Graffe, S. Blanquet, and M. Grunberg-Manago. 1985. Attenuation control of the Escherichia coli phenylalanyl tRNA synthetase operon. J. Mol. Biol. 181:467–478.
305. Springer, M., M. Trudel, M. Graffe, J. A. Plumbridge, G. Fayat, J. F. Mayaux, C. Sacerdot, S. Blanquet, and M. Grunberg-Manago. 1983. Escherichia coli phenylalanyl-tRNA synthetase operon is controlled by attenuation "in vivo." J. Mol. Biol. 171:263–279.
306. Srinivasan, P. R., M. Katagiri, and D. B. Sprinson. 1959. The conversion of phosphoenolpyruvic acid and d-erythrose 4-phosphate to 5-dehydroquinic acid. J. Biol. Chem. 234:713–715.
307. Srinivasan, P. R., J. Rothschild, and D. B. Sprinson. 1963. The enzymic conversion of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate to 5-dehydroquinate. J. Biol. Chem. 238:3176–3182.
308. Srinivasan, P. R., H. T. Shigeura, M. Sprecher, D. B. Sprinson, and B. D. Davis. 1956. The biosynthesis of shikimic acid from d-glucose. J. Biol. Chem. 220:447–497.
309. Srinivasan, P. R., and D. B. Sprinson. 1959. 2-Keto-3-deoxy-d-arabino-heptonic acid 7-phosphate synthetase. J. Biol. Chem. 234:716–722.
310. Staacke, D., B. Walter, B. Kisters-Wolke, B. vonWicken-Bergmann, and B. Müller-Hill. 1990. How Trp-repressor binds to its operator. EMBO J. 9:1963–1967.
311. Stalker, D., W. Hiatt, and L. Comai. 1985. A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. J. Biol. Chem. 260:4724–4728.
312. Staub, M., and S. Denes. 1969. Purification and properties of the 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K-12. Biochim. Biophys. Acta 178:588–598.
313. Tamir, H., and P. R. Srinivasan. 1969. Purification and properties of anthranilate synthase from Salmonella typhimurium. J. Biol. Chem. 224:6507–6513.
314. Tribe, D. E., H. Camakaris, and J. Pittard. 1976. Constitutive and repressible enzymes of the common pathway of aromatic biosynthesis in Escherichia coli K-12: regulation of enzyme synthesis at different growth rates. J. Bacteriol. 127:1085–1097.
315. Tribe, D. E., and J. Pittard. 1979. Hyperproduction of tryptophan by Escherichia coli: genetic manipulation of the pathways leading to tryptophan formation. Appl. Environ. Microbiol. 38:181–190.
316. Tschopp, J., and K. Kirschner. 1980. Subunit interactions of tryptophan synthase from Escherichia coli as revealed by binding studies with pyridoxal phosphate analogues. Biochemistry 19:4514–4521.
317. Tschopp, J., and K. Kirschner. 1980. Kinetics of cooperative ligand binding to the apoβ2 subunit of tryptophan synthase and its modulation by the α subunit. Biochemistry 19:4522–4526.
318. Umbarger, H. E., and J. H. Mueller. 1951. Isoleucine and valine metabolism of Escherichia coli. 1. Growth studies on amino acid-deficient mutants. J. Biol. Chem. 189:277–285.
319. Vaz, A. D. N., J. R. Butler, and M. J. Nugent. 1975. Dehydroquinase catalyzed dehydration. II. Identification of the reactive conformation of the substrate responsible for syn elimination. J. Am. Chem. Soc. 97:5914–5915.
320. Wallace, B.J., and J. Pittard. 1967. Genetic and biochemical analysis of the isoenzymes concerned in the first reaction of aromatic biosynthesis in Escherichia coli. J. Bacteriol. 93:237–244.
321. Wallace, B. J., and J. Pittard. 1967. Chromatography of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate synthetase (Trp) on diethylaminoethyl cellulose: a correction. J. Bacteriol. 94:1279–1280.
322. Wallace, B. J., and J. Pittard. 1969. Regulator gene controlling enzymes concerned in tyrosine biosynthesis in Escherichia coli. J. Bacteriol. 97:1234–1241.
323. Wallace, B. J., and J. Pittard. 1969. Regulation of 3-deoxy-d-arabino-heptulosonic-7-phosphate acid synthetase activity in relation to the synthesis of the aromatic vitamins in Escherichia coli K-12. J. Bacteriol. 99:707–712.
324. Weaver, L. M., and K. M. Herrmann. 1990. Cloning of an aroF allele encoding a tyrosine insensitive 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase. J. Bacteriol. 172:6581–6584.
325. Weiss, D. S., J. Batut, K. E. Klose, J. Keener, and S. Kustu. 1991. The phosphorylated form of the enhancer-binding protein NtrC has an ATPase activity that is essential for activation of transcription. Cell 67:155–167.
326. Weiss, U., B. D. Davis, and E. S. Mingioli. 1953. Aromatic biosynthesis. X. Identification of an early precursor as 5-dehydroquinic acid. J. Am. Chem. Soc. 75:5572–5576.
327. Weiss, U., C. Gilvarg, E. S. Mingioli, and B. D. Davis. 1954. Aromatic biosynthesis. XI. The aromatization step in the synthesis of phenylalanine. Science 119:774–775.
328. Weiss, U., and E. S. Mingioli. 1955. Aromatic biosynthesis. XV. The isolation and identification of shikimic acid 5-phosphate. J. Am. Chem. Soc. 78:2894–2898.
329. Whipp, M. J., D. M. Halsall, and A. J. Pittard. 1980. Isolation and characterization of an Escherichia coli K-12 mutant defective in tyrosine- and phenylalanine-specific transport systems. J. Bacteriol. 143:1–7.
330. Whipp, M. J., and A. J. Pittard. 1977. Regulation of aromatic amino acid transport systems in Escherichia coli K-12. J. Bacteriol. 132:453–461.
330a. Whipp, M. J., and A. J. Pittard. 1995. A reassessment of the relationship between aroK- and aroL-encoded shikimate kinase enzymes of Escherichia coli. J. Bacteriol. 177:1627–1629.
331. White, J., G. Millar, and J. R. Coggins. 1988. The overexpression purification and complete amino acid sequence of chorismate synthase from Escherichia coli K-12 and its comparison with the enzyme from Neurospora crassa. Biochem. J. 251:313–322.
332. White, J. L., M. G. Grutter, E. Wilson, C. Thaller, G. C. Ford, J. D. G. Smit, J. N. Jansonius, and K. Kirschner. 1982. Crystallization and preliminary X-ray crystallographic data of the bifunctional enzyme phosphoribosyl-anthranilate isomerase indole-3-glycerol phosphate synthase from Escherichia coli. FEBS Lett. 148:87–90.
333. Williams, M. V., T. J. Kerr, R. D. Lemmon, and G. J. Tritz. 1980. Azaserine resistance in Escherichia coli: chromosomal location of multiple genes. J. Bacteriol. 143:383–388.
334. Wilson, D. A., and I. P. Crawford. 1965. Purification and properties of the B component of Escherichia coli tryptophan synthetase. J. Biol. Chem. 240:4801–4808.
335. Wilson, R. K., T. Brown, and B. A. Roe. 1986. Nucleotide sequence of pheW: a third gene for Escherichia coli tRNAPhe. Nucleic Acids Res. 14:5937.
336. Wilson, T. J., P. Maroudas, S. J. Howlett, and B. E. Davidson. 1994. Ligand-induced self-association of the Escherichia coli regulatory protein TyrR. J. Mol. Biol. 238:309–318.
337. Wookey, P. J., J. Pittard, S. M. Forrest, and B. E. Davidson. 1984. Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12. J. Bacteriol. 160:169–174.
338. Yang, J., H. Camakaris, and A. J. Pittard. 1993. Mutations in the tyrR gene of Escherichia coli which affect TyrR-mediated activation but not TyrR-mediated repression. J. Bacteriol. 179:6372–6375.
339. Yang, J., S. Ganesan, J. Sarsero, and A. J. Pittard. 1993. A genetic analysis of various functions of the TyrR protein of Escherichia coli. J. Bacteriol. 175:1767–1776.
340. Yang, J., and A. J. Pittard. 1987. Molecular analysis of the regulatory region of the Escherichia coli K-12 tyrB gene. J. Bacteriol. 169:4710–4715.
341. Yang, X. Y., and E. W. Miles. 1992. Threonine 183 and adjacent flexible loop residues in tryptophan synthase α subunit have critical roles in modulating the enzymatic activities of the β subunit in the α2β2 complex. J. Biol. Chem. 267:7520–7528.
342. Yaniv, H., and C. Gilvarg. 1955. Aromatic biosynthesis. XIV. 5-Dehydroshikimic reductase. J. Biol. Chem. 213:787–795.
343. Yanofsky, C. 1956. The enzymic conversion of anthranilic acid to indole. J. Biol. Chem. 223:171–185.
344. Yanofsky, C. 1959. A second reaction catalyzed by the tryptophan synthetase of Escherichia coli. Biochim. Biophys. Acta 31:409–416.
345. Yanofsky, C. 1960. The tryptophan synthetase system. Bacteriol. Rev. 24:221–245.
346. Yanofsky, C. 1981. Attenuation in the control of expression of bacterial operons. Nature (London) 289:751–758.
347. Yanofsky, C., and I. P. Crawford. 1972. Tryptophan synthetase, p. 1–31. In P. D. Boyer (ed.), The Enzymes, vol. VII, 3rd ed. Academic Press, Inc., New York.
348. Yanofsky, C., G. Drapeau, J. R. Guest, and B. C. Carlton. 1967. The complete amino acid sequence of the tryptophan synthetase A protein (α subunit) and its colinear relationship with the genetic map of the A gene. Proc. Natl. Acad. Sci. USA 57:296–298.
349. Yanofsky, C., V. Horn, M. Bonner, and S. Stasiowski. 1971. Polarity and enzyme functions in mutants of the first three genes of the tryptophan operon of Escherichia coli. Genetics 69:409–433.
350. Yanofsky, C., V. Horn, and P. Gollnick. 1991. Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli. J. Bacteriol. 173:6009–6117.
351. Yanofsky, C., and E. S. Lennox. 1959. Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli. Virology 8:425–447.
352. Yanofsky, C., J. Platt, I. P. Crawford, B. P. Nichols, G. E. Christie, H. Horowitz, M. Van Cleemput, and A. M. Wu. 1981. The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 9:6647–6668.
353. Yanofsky, C., and M. Rachmeler. 1958. The exclusion of free indole as an intermediate in the biosynthesis of tryptophan in Neurospora crassa. Biochim. Biophys. Acta 28:640–641.
354. Yanofsky, C., and J. Stadler. 1958. The enzymatic activity associated with the protein immunologically related to tryptophan synthetase. Proc. Natl. Acad. Sci. USA 44:245–253.
355. Yanosky, C., and M. van Cleemput. 1982. Nucleotide sequence of trpE of Salmonella typhimurium and its homology with the corresponding sequence of Escherichia coli. J. Mol. Biol. 155:235–246.
356. Zalkin, H. 1973. Anthranilate synthetase. Adv. Enzymol. 38:1–37.
357. Zhang, H., D. Zhao, C. Arrowsouth, and O. Jardetsky. 1994. The solution structure of trp repressor-operator DNA complex. J. Mol. Biol. 238:592–614.
358. Zhang, R. S., A. Joachimiak, C. L. Lawson, R. W. Schevitz, Z. Otwinowski, and P. B. Sigler. 1987. The crystal structure of trp aporepressor at 1.8 Å shows how binding of tryptophan enhances DNA affinity. Nature (London) 327:591–597.
359. Zurawski, G., K. Brown, D. Killingly, and C. Yanofsky. 1978. Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4271–4275.
360. Zurawski, G., R. P. Gunsalus, K. D. Brown, and C. Yanofsky. 1981. Structure and regulation of aroH, the structural gene for the tryptophan-repressible 3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthetase of Escherichia coli. J. Mol. Biol. 145:47–73.
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