Chapter 41

Folate Biosynthesis, Reduction, and Polyglutamylation

JACALYN M. GREEN, BRIAN P. NICHOLS, and ROWENA G. MATTHEWS

INTRODUCTION

Folic acid derivatives serve as one-carbon donors in a wide variety of cellular reactions. Tetrahydrofolate serves as a recipient for one-carbon units generated during glycine cleavage to form CO₂ and NH₃ and during the biosynthesis of glycine from serine. 5,10-Methylenetetrahydrofolate serves as a one-carbon donor for the methylation of dUMP to form dTMP and also in the biosynthesis of pantothenate. 10-Formyltetrahydrofolate is the one-carbon donor in purine biosynthesis and for the formylation of fMet-tRNA, and 5-methyltetrahydrofolate provides one-carbon units for methionine biosynthesis. Folate derivatives also participate in other cellular processes that do not involve one-carbon transfers. Methenyltetrahydrofolate plays a role in the repair of pyrimidine dimers by DNA photolyase (29), and dihydrofolate polyglutamate is involved in the assembly of the baseplate of T4 phage.

In contrast to humans, for whom folate is a dietary requirement, many microorganisms and plants possess the ability to synthesize folic acid derivatives de novo. The end product of the de novo biosynthesis of folic acid is dihydrofolate. This folic acid derivative is not active in one-carbon transfer reactions and must first be reduced to the level of tetrahydrofolate. All of the folic acid derivatives that serve as recipients and donors of one-carbon units are derivatives of tetrahydrofolate, which is formed from dihydrofolate by an NADPH-dependent reduction catalyzed by dihydrofolate reductase (Fig. 1).

Fig. 1Structures of 7,8-dihydrofolate (dihydrofolate, H2folate) and 5,6,7,8-tetrahydrofolate (tetrahydrofolate, H4folate). The R group in each of these structures is the benzoylglutamic acid moiety of p-aminobenzoylglutamic acid.

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The product of de novo dihydrofolate biosynthesis is also a monoglutamate; the substituent at C-6 is p-aminobenzoylmonoglutamate. However, the intracellular forms of folic acid are almost entirely polyglutamates, formed by addition of two to seven glutamyl residues to the monoglutamate. The polyglutamate "tail" is required for intracellular retention of folic acid derivatives and plays an important role in the binding of folate cosubstrates to enzymes involved in one-carbon transfer or interconversion of folic acid derivatives (49). Most folate-dependent enzymes use both monoglutamate and polyglutamate forms of folic acid derivatives as substrates, but polyglutamate derivatives are usually better substrates, with lower Michaelis constants (K_m values) and/or with higher V _max values. However, the cobalamin-independent methionine synthase encoded by the metE gene shows an absolute requirement for polyglutamates of methyltetrahydrofolate (78). Strains of Escherichia coli with greatly reduced folylpolyglutamate synthetase activity are auxotrophic for methionine or vitamin B₁₂ (vitamin B₁₂ supplementation permits methionine synthesis using the cobalamin-dependent MetH protein, which can use methyltetrahydrofolate monoglutamate as a substrate), and their growth is stimulated by addition of glycine and thymine to the medium (8).

In this chapter, we will discuss the biosynthesis of dihydrofolate monoglutamate, its reduction to tetrahydrofolate monoglutamate, and the addition of glutamyl residues to form folylpolyglutamates. The generation of the active cosubstrates methyl-, methylene-, and formyltetrahydrofolate from tetrahydrofolate and their use in one-carbon transfer reactions are discussed in chapter 36. E. coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium), like many microorganisms that can synthesize folate de novo, appear to lack the ability to transport folate into the cell and are thus highly susceptible to inhibitors of folate biosynthesis. This chapter will include a brief discussion of inhibition of folate biosynthesis by sulfa drugs.

BIOSYNTHESIS OF DIHYDROFOLATE

The folate biosynthetic pathway can be divided into parts. First, the aromatic precursor chorismate is converted to p-aminobenzoic acid (PABA) by the action of three proteins. Second, the pteridine portion of folate is made from GTP, in a series of reactions that requires six enzymes. This portion of folate biosynthesis was extensively reviewed in the first edition of this book (10), which can be referred to for a more historical perspective. Next, PABA and 6-hydroxymethyl-7,8-dihydropterin (6-CH_2OH-H₂pterin) pyrophosphate are coupled to form dihydropteroate (H₂pteroate) by H₂pteroate synthase, and the bifunctional protein encoded by folC, dihydrofolate synthetase/folylpolyglutamate synthetase, adds the initial glutamate molecule to form dihydrofolate (dihydropteroylmonoglutamate [H₂PteGlu₁]). Dihydrofolate is then reduced to tetrahydrofolate by dihydrofolate reductase (FolA), and the folylpolyglutamate synthetase function of FolC adds two additional glutamyl residues to form tetrahydropteroyltriglutamate (H₄PteGlu₃). The overall synthesis of tetrahydrofolate is shown in Fig. 2 along with the structures of the intermediates. The chromosomal locations of genes known to be involved in folate biosynthesis are also shown in Fig. 2. Since folate and its reduced derivatives cannot cross the inner membrane of E. coli, most of the folate biosynthetic pathway was first worked out biochemically by isolating intermediates and characterizing purified or partially purified proteins; much of this early work was accomplished in the laboratory of Gene Brown. In contrast, since PABA does freely enter E. coli cells, this part of the pathway was first identified genetically, with the biochemistry being completed later.

Fig. 2Sequence of reactions involved in the biosynthesis of dihydrofolic acid from GTP and chorismate. The prefixes H2 and H4 denote dihydro and tetrahydro, respectively.

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Biosynthesis of PABA from Chorismate

Although it was shown in 1950 that PABA derived from the same pathway as the aromatic amino acids tyrosine, phenylalanine, and tryptophan, it was only in 1967 that Huang and Pittard (33) used mutagenesis techniques to obtain strains of E. coli that required PABA for growth. They identified two loci, which they named pabA and pabB. In 1983, Kaplan and Nichols (36) cloned pabA. Nucleotide sequence analysis of the gene revealed the deduced amino acid sequence to be 44% identical with that of anthranilate synthase component II. pabB was cloned shortly thereafter, and sequence analysis of the predicted polypeptide gene product revealed a 26% similarity to the trpE gene product, which encodes component I of anthranilate synthase (23). Despite this similarity, the subunits of anthranilate synthase could not substitute for the analogous subunits in PABA synthase or vice versa.

In 1989, Nichols et al. (51) constructed two plasmid-containing strains that overexpressed the gene products of pabA and pabB. Studies with these partially purified enzymes showed that the conversion of chorismate to PABA occurred in two steps. PabA and PabB acted upon chorismate and glutamine to form a diffusible intermediate, which was then converted to PABA by the action of a third enzyme, at that time termed enzyme X. In 1985, Teng et al. (73) synthesized the proposed intermediate in the biosynthesis of PABA, 4-amino-4-deoxychorismate (ADC) (Fig. 2). They found that cell extracts from a strain overexpressing PabB converted ADC to PABA at a rate commensurate with a biological function for this compound as an intermediate in PABA biosynthesis. This intermediate was subsequently identified as ADC (2, 80). Enzyme X was then renamed ADC lyase, and the complex containing PABA synthase components I and II, the pabA and pabB gene products, was named ADC synthase.

In summary, PABA is one of seven aromatic products derived from chorismate in E. coli. Chorismate is converted to PABA in two steps through the action of three gene products, PabA, PabB, and PabC. ADC synthase, a heterodimer of PabA and PabB, converts chorismate and glutamine to ADC and glutamate. ADC lyase aromatizes ADC, releasing pyruvate and generating PABA.

Although the early literature is replete with references to PABA synthase, it is likely that these studies involved ADC synthase components I and II, with enough contaminating lyase to support activity, since gel filtration experiments fail to separate the native synthase from the lyase, both of which elute with apparent molecular weights of about 50,000 (56).

ADC Synthase (PabA, PabB).

ADC synthase or its components can catalyze the following reactions:

chorismate + glutamine → ADC + glutamate  (1)

chorismate + NH₃ ↔ ADC  (2)

glutamine → glutamate + NH₃  (3)

Although reactions 1 and 3 require both ADC synthase components I and II, the gene products of pabB and pabA, respectively, reaction 2 can be carried out by PabB alone.

Current research suggests that the physiological reaction occurs as shown below:

PabA acts as a glutaminase that generates ammonia from glutamine, although activation requires equimolar amounts of PabB (63). PabB then uses the ammonia to aminate chorismate, generating ADC (63). PabC acts separately to generate PABA from ADC (26). Research by Viswanathan strongly suggests that free ammonia is not a physiological substrate for PabB (77). For reaction 2, the measured K_m value for ammonia was 360 mM in the presence of PabA and 140 mM in the absence of PabA, whereas the K_m value for glutamine in reaction 1 was about 1 mM. pabA mutants require more than 50 mM ammonia to grow (E. Rayl and B. P. Nichols, unpublished data). Hydrolysis of cellular glutamine will not generate enough ammonia to support the PabB-dependent amination of chorismate. In contrast, trpG mutants, which lack anthranilate synthase component II, require only 1 mM ammonia for growth (82).

A recent study by Rayl defined conditions under which PabA and PabB are associated (56). The ADC synthase complex is probably an αβ dimer. PabA and PabB are associated most tightly when preincubated at 37°C for 90 min in the presence of 5 mM glutamine. PabC does not appear to form a complex with PabA and PabB (56).

The pabA genes from E. coli and S. typhimurium have been shown to specify polypeptides with a predicted molecular mass of 21 kDa (35, 36). Expression studies reveal that pabA from E. coli is expressed constitutively from a monocistronic transcript (74, 75). However, it has also been reported that pabA is expressed from a transcript including fic, the gene upstream of pabA (40).

The pabB genes from E. coli (23) and S. typhimurium (24) have been isolated, and their nucleotide sequences have been analyzed. The pabB gene consists of 1,359 nucleotides specifying a protein of 453 residues with a predicted molecular mass of ∼51 kDa.

ADC Lyase (PabC).

ADC lyase catalyzes the elimination of pyruvate from ADC and the aromatization of the resulting product to generate PABA. The enzyme was identified in 1989 (51), partially purified in 1990 (80), and further purified in 1991 (26). The enzyme was shown to be a homodimer of ∼25-kDa subunits (26, 51, 80). Sequence analysis of the cloned pabC gene identified an open reading frame specifying a protein of 29,700 Da (25). The purified, overexpressed protein was shown to contain a pyridoxal phosphate cofactor (25).

Transport of PABA.

Little is known about the transport of PABA, although it is clear that PABA is transported and can support the growth of strains unable to synthesize PABA from chorismate (75). In fact, PABA is excreted from wild-type cells and can support the growth of strains that are PABA auxotrophs (33).

Biosynthesis of Dihydropteroic Acid from GTP and PABA

As shown in Fig. 2, the synthesis of dihydropteroic acid uses GTP as the initial substrate and requires the sequential action of GTP cyclohydrolase I, dihydroneopterin pyrophosphatase, a phosphomonoesterase, dihydroneopterin aldolase, 6-CH₂OH-H₂pterin pyrophosphokinase, and H₂pteroate synthase. Figure 2 also provides the structural gene designation and physical map position where known.

GTP Cyclohydrolase I (FalE).

GTP cyclohydrolase I, so named to differentiate this enzyme from GTP cyclohydrolase II, which catalyzes the first step in the biosynthesis of riboflavin from GTP, was identified in E. coli and subsequently characterized by Brown and coworkers (12, 21, 79). The enzyme catalyzes the following overall reaction:

GTP + 2H₂O → dihydroneopterin triphosphate + formate

The reaction occurs in four steps, which include an Amadori rearrangement, and the intermediates are probably enzyme bound (Fig. 3).

Fig. 3Reactions and enzyme-bound intermediates involved in the conversion of GTP to dihydroneopterin (H2neopterin) triphosphate in the presence of the enzyme GTP cyclohydrolase. Structures I, II, and III are enzyme-bound intermediates.

Source: Reprinted with permission from reference 10.

The enzyme was purified 3,900-fold to homogeneity by Yim and Brown (81). The enzyme is a tetramer of dimers, with a subunit molecular mass of ∼25 kDa. It binds eight molecules of GTP and has a K_m of 20 nM for GTP. Unlike many other enzymes that catalyze the committing step in a biochemical pathway, GTP cyclohydrolase I exhibits no feedback inhibition by end products.

The gene coding for E. coli GTP cyclohydrolase I, folE, has been cloned (37), the sequence has been determined (38), and the gene has been mapped at 45 min on the E. coli chromosome (∼2,250 kbp) (59). The open reading frame contains 669 nucleotides, encoding a protein with a predicted molecular weight of 25,873.

Dihydroneopterin Triphosphate Pyrophosphatase.

Suzuki and Brown (67) determined that two separate enzyme-catalyzed steps were necessary in the conversion of dihydroneopterin triphosphate to dihydroneopterin. The first step is catalyzed by dihydroneopterin triphosphate pyrophosphatase, and the removal of the third phosphate group is catalyzed by nonspecific phosphate monoesterases.

E. coli dihydroneopterin triphosphate pyrophosphatase catalyzes the removal of pyrophosphate from dihydroneopterin triphosphate to yield dihydroneopterin monophosphate. This enzyme was purified ∼50-fold and partially characterized by Suzuki and Brown (67). The molecular weight of the enzyme is 17,000. The enzyme requires magnesium ion for activity and will not recognize nucleoside triphosphates as substrates. The K_m value for dihydroneopterin triphosphate is 11 μM. The gene encoding dihydroneopterin triphosphate pyrophosphatase has not yet been cloned.

Phosphomonoesterase.

Suzuki and Brown (67) showed that several phosphomonoesterases in E. coli could catalyze the removal of the phosphate group from dihydroneopterin monophosphate, but no single enzyme showed specificity for dihydroneopterin monophosphate.

Dihydroneopterin Aldolase.

Dihydroneopterin aldolase reacts with dihydroneopterin to generate 6-CH₂OH-H₂pterin and glycolaldehyde. The enzyme from E. coli is remarkably heat stable, maintaining full activity even after 5 min at 100°C, which greatly aided in purification of the enzyme (48). The K_m value for dihydroneopterin is 9 μM. The enzyme is highly specific for dihydroneopterin and will not use neopterin or the mono- or triphosphate of dihydroneopterin. The product 6-CH₂OH-H₂pterin is a good inhibitor, with a K_i of 1.7 μM. The gene encoding this protein has not yet been cloned.

6-CH₂OH-H2_pterin Pyrophosphokinase (FolK).

6-CH₂OH-H₂pterin pyrophosphokinase converts 6-CH₂OH-H₂pterin to its pyrophosphate ester, with ATP as the donor of the pyrophosphate group (57). The enzyme activity requires Mg²⁺. Talarico et al. (70) have purified 6-CH₂OH-H₂pterin pyrophosphokinase more than 10,000-fold to homogeneity. The native enzyme had a mass of 25 kDa as determined by gel electrophoresis in the presence and absence of sodium dodecyl sulfate and thus must be monomeric. Kinetic studies with the pure protein yielded K_m values of 1.6 μM for 6-CH₂OH-H₂pterin and 17 μM for ATP.

The folK gene was subsequently cloned, its sequence was determined, and the gene product was overexpressed (71). The open reading frame of the folK gene was 477 bp in length, coding for a protein with a predicted molecular weight of 17, 945. The open reading frame for folK is just 8 nucleotides downstream of pcnB, a gene involved in controlling plasmid copy number (46), and pcnB and folK may be transcribed as a polycistronic mRNA.

H2pteroate Synthase (FalP and R-Factor-Encoded Enzyme).

H₂pteroate synthase catalyzes the condensation of PABA and 6-CH₂OH-H₂pterin pyrophosphate to form H₂pteroate. The reactants and products in this reaction in E. coli were elucidated by Richey and Brown (57). Although p-aminobenzoylglutamate (PABAGlu) could be used instead of PABA as the substrate to generate dihydrofolate, H₂pteroate synthase used PABA 10 times more efficiently than PABAGlu. For the purified enzyme from E. coli , the K_m for PABA is 2.5 μM, whereas the K_i for PABAGlu as a competitive inhibitor is 1.3 mM (58). This observation, together with the fact that it proved impossible to isolate a PABAGlu-requiring strain and no enzyme was ever found in any organism that could condense PABA and glutamate, led researchers to conclude that the biological substrate for H₂pteroate synthase was PABA and that H₂pteroate was the true intermediate in the synthesis of dihydrofolate.

Talarico et al. (70) purified H₂pteroate synthase more than 700-fold from a wild-type strain, obtaining apparently homogeneous enzyme. The native enzyme is a dimer of ∼30-kDa subunits. Kinetic studies of the pure enzyme yielded K_m values for 6-CH₂OH-H₂pterin pyrophosphate and PABA of 1.9 and 0.5 μM, respectively.

Dallas et al. (14, 15) cloned, determined the sequence of, and overexpressed the folP gene, encoding H₂pteroate synthase, which lies at 71.5 min. The open reading frame of the gene contains 846 bp and is predicted to code for a polypeptide of 282 amino acids with a mass of 30,314 Da.

Biosynthesis of 7,8-Dihydrofolate

Dihydrofolate synthetase catalyzes the synthesis of dihydrofolate from H₂pteroate, glutamate, and ATP and requires Mg²⁺ for activity. The enzyme was partially purified, and the reaction was first characterized in E. coli by Griffin and Brown (27). In E. coli, dihydrofolate synthetase and folylpolyglutamate synthetase are activities contained on a single bifunctional protein (17, 19). In 1985, Bognar et al. cloned the folC gene and overexpressed the folC gene product ∼400-fold over levels in a wild-type strain (8). They purified the overexpressed dihydrofolate synthetase activity to homogeneity. Dihydrofolate synthetase activity copurified with folylpolyglutamate synthetase activity. The enzyme was shown to be a monomer of ∼45 kDa. Kinetic experiments were performed, and the K_m values for H₂pteroate, ATP, and glutamate were found to be 0.6 μM, 6.9 μM, and 3.9 mM respectively.

The folC gene is essential in E. coli, as evidenced by the observation that the chromosomal gene can be inactivated only if a second folC gene is present in trans (54). The availability of strains with folC deleted from the chromosome permitted the isolation and characterization of plasmid-encoded mutant folC gene products in the absence of wild-type enzyme. Such mutant genes are described in reports from the Bognar laboratory (39, 54).

Intervention by Sulfa Drugs

Sulfonamide derivatives are structural analogs of PABA, and they compete with PABA for condensation with 6-hydroxymethylpterin pyrophosphate in the reaction catalyzed by H₂pteroate synthase (9, 61) (Fig. 4). The pterin-sulfonamide adducts are thought not to be inhibitory to cellular function, and they passively diffuse out of the cell (61). Since sulfonamide derivatives compete with PABA for H₂pteroate synthase, the cell becomes depleted in H₂pteroate and reduced folates derived from H₂pteroate (6, 31, 61). Mechanisms of development of sulfonamide resistance include alterations in cellular transport properties (9, 34, 53), modification of the inhibitor (9, 61), overproduction of PABA (11; G. G. Guay and B. P. Nichols, unpublished data), and mutation of H₂pteroate synthase.

Fig. 4Comparison of the structures of sulfonamide and PABA.

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The mechanism of sulfonamide resistance that has been best characterized is mutation to form a sulfonamide-resistant H₂pteroate synthase. Several groups have isolated resistant H₂pteroate synthases from strains of E. coli that are resistant to sulfathiazole (15, 53, 68; G. Vedantam and B. P. Nichols, unpublished data). In all cases so far characterized, the resistant H₂pteroate synthase had a decreased ratio of the K_m for PABA over the K_i for sulfanilamide, allowing the enzyme to discriminate more effectively between the natural substrate and the inhibitor (15, 68). In two cases, the mutant folP has been sequenced and found to contain a Phe-28-to-Leu change (15) or a Pro-64-to-Ser mutation (Vedantam and Nichols, unpublished data). In addition, sulfonamide resistance conferred by R plasmids isolated from resistant pathological strains of E. coli have been shown to contain resistant H₂pteroate synthases (72). Two different H₂pteroate synthase genes, sulI and sulII, have been identified from R plasmids (69). Sequence analysis revealed that these genes share 50% identity but are not closely related to folP from the E. coli chromosome (28, 55, 66).

REDUCTION AND POLYGLUTAMYLATION OF FOLIC ACID DERIVATIVES

Reduction of Dihydrofolate to Tetrahydrofolate

The chromosomally encoded gene for dihydrofolate reductase in E. coli (folA) encodes a monomeric protein of ∼18,000 Da. This protein is very sensitive to inhibition by trimethoprim and its analogs, forming the basis for effective treatments directed against gram-negative organisms. Resistance to trimethoprim is often associated with the production of a plasmid-encoded dihydrofolate reductase that is structurally unrelated and is a tetramer of ∼8,000-Da subunits.

Dihydrofolate Reductase (FolA).

The major chromosomally encoded enzyme responsible for the reduction of dihydrofolate to the physiologically active tetrahydrofolate is the folA gene product. The enzyme dihydrofolate reductase catalyzes the following reaction:

NADPH + H⁺ + dihydrofolate ↔ NADP⁺ + tetrahydrofolate

Although dihydrofolate reductases from many organisms, including some strains of E. coli, will also carry out a very slow reduction of folic acid to dihydrofolate (7), strains of E. coli do not transport folates, and this reaction presumably does not occur in vivo. The E. coli enzyme is needed to reduce newly synthesized dihydrofolate to tetrahydrofolate. Dihydrofolate is also formed in the reaction catalyzed by thymidylate synthase:

dUMP + methylenetetrahydrofolate → dTMP + dihydrofolate

In this reaction, methylenetetrahydrofolate serves a dual function, providing both a one-carbon unit (the methylene group) for the methylation of dUMP, and the reducing equivalents necessary to convert a methylene to a methyl group. Dihydrofolate reductase must therefore reduce dihydrofolate as rapidly as it is formed during the de novo biosynthesis of dTMP in order to prevent the depletion of cellular stores of tetrahydrofolate derivatives.

Since E. coli dihydrofolate reductase is a rather small protein of considerable importance as a target for chemotherapy, it has been extensively characterized, and its structure is known to high resolution. Excellent reviews and original papers can be consulted for detailed information about the structure and mechanism of the enzyme (7, 13, 20).

The enzyme is usually assayed spectrophotometrically by measuring the consumption of NADPH at 340 nm during reduction of dihydrofolate to tetrahydrofolate (47). The absorbance changes at 340 nm are due both to the oxidation of NADPH and to the reduction of dihydrofolate and are associated with a molar extinction change at 340 nm of 12,300 (7). This method is not suitable for measurement of activity in crude extracts, for which a radioisotopic method of activity measurement should be used (60).

The wild-type chromosomal gene for dihydrofolate reductase (folA) in an E. coli K-12 strain was cloned into pBR322 following its enrichment by bacteriophage Mu-mediated transposition (62). The predicted amino acid sequence was 160 residues long, and the predicted molecular weight of the polypeptide is 17,998. folA lies at 1 min on the E. coli chromosome (5).

Although it might have been expected that a deletion mutation leading to complete loss of folA expression would be lethal, strains containing a deletion of the folA gene have recently been isolated from thyA strains independently in two different laboratories (1, 32). These strains are auxotrophic for thymine, adenine, pantothenate, glycine, and methionine and are viable only in the thyA background. The auxotrophies are those expected for a strain that is unable to produce sufficient reduced forms of tetrahydrofolate for one-carbon transfer reactions. The requirement for a thyA background is more perplexing. thyA is the structural gene for thymidylate synthase, and a thyA mutant would therefore fail to generate dihydrofolate from tetrahydrofolate. However, if there were no tetrahydrofolate derivatives in strains lacking folA, thymidylate synthase activity should not affect the phenotype. A solution to this dilemma appeared with the observations of Hamm-Alvarez et al. (30) that tetrahydrofolate derivatives are present in the strains with deletions of the folA gene. Although the identity of the enzyme responsible for formation of tetrahydrofolate derivatives in folA strains is not known, it may be the recently identified dihydropteridine reductase of E. coli (3, 76), which has dihydrofolate reductase activity.

R-Plasmid-Encoded Dihydrofolate Reductase.

Shortly after trimethoprim was introduced as a clinical drug, bacterial strains that were resistant to trimethoprim and contained an R plasmid specifying a trimethoprim-insensitive dihydrofolate reductase were isolated (4). Two types of resistant dihydrofolate reductase appeared to be synthesized: type I enzyme, exhibiting a several- thousand-fold reduction in sensitivity to trimethoprim, and type II enzyme, with complete insensitivity to trimethoprim. The type II enzymes are especially interesting because they are genetically and structurally unrelated to chromosomal dihydrofolate reductases. The R67 type II enzyme is a tetramer of identical 8.4-kDa subunits (65), and the active site is located in a pore passing through the center of the protein, with residues from each monomer contributing the the active site (83). The evolutionary origin of the R plasmids is not known.

Formation of Folylpolyglutamates

Folylpolyglutamate Synthetase/Dihydrofolate Synthase (FolC).

The monoglutamate form of dihydrofolate that is the product of the de novo biosynthetic pathway discussed above is a substrate for dihydrofolate reductase, and the tetrahydrofolate monoglutamate product is itself a substrate for serine hydroxymethyltransferase and the enzymes that interconvert methylenetetrahydrofolate with other tetrahydrofolate cofactors. The addition of glutamyl residues by folylpolyglutamate synthetase probably occurs after the reduction of newly synthesized dihydrofolate to tetrahydrofolate and its conversion to other tetrahydrofolate derivatives. The folC gene product is a bifunctional enzyme that also catalyzes the addition of the glutamyl residue to H₂pteroate to form dihydrofolate (see above). The two reactions catalyzed by FolC are as follows:

H₂pteroate + ATP + l-glutamate → tetrahydrofolate (H₂PteGlu₁) + ADP + HPO₄ ^2–

H₄PteGlu_n + ATP + l-glutamate → H₄PteGlu_{n +1} + ADP + HPO₄ ^2–

FolC adds successive glutamyl residues to H₄PteGlu₁ by linking each glutamyl residue to the γ-carboxyl group of the preceding glutamyl residue (Fig. 5). Thus, the isopeptide bonds formed are not the normal amide bonds to the α-carboxyl of glutamate and are not hydrolyzed by peptidases or proteases that are specific for α-carboxyl-linked peptide bonds. The preferred monoglutamate substrate is 10-CHO-H₄PteGlu₁, and H₂PteGlu₁ is a very poor substrate (B. Shane, personal communication). The preferred diglutamate substrate is 5,10-CH₂-H₄PteGlu₂. Although the enzyme forms triglutamates and tetraglutamates, formation of the longer-chain-length folates occurs very slowly.

Fig. 5The structure of tetrahydropteroylpentaglutamate from E. coli. This pentaglutamate form of tetrahydrofolate contains two glutamyl residues linked via the ?-carboxyls of the preceding glutamyl residue and two glutamyl residues linked via the ?-carboxyl of the preceding glutamyl residue, as indicated in the structure. As noted in the text, the synthesis of the pentaglutamate derivative is thought to be mediated by two different folylpolyglutamate synthase enzymes.

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A Folylpolyglutamate Synthetase That Forms α-Linked Peptide Bonds.

Studies on the in vivo distribution of polyglutamate chain lengths of folate derivatives in E. coli during exponential growth in LB (22) indicated that mono- and triglutamate derivatives were most abundant, with tetra-, penta-, and hexaglutamate derivatives also found in order of decreasing abundance. However, in stationary phase, cells were found to contain longer-chain-length folypolyglutamates, with the predominant chain length now containing six or seven glutamyl residues. Since the FolC enzyme predominantly forms triglutamyl residues both in vivo (52) and in vitro (Shane, personal communication), the in vivo distribution of folylpolyglutamates was puzzling. Ferone and colleagues (16, 18) have shown that there is a second folylpolyglutamate synthase in E. coli that adds additional glutamyl residues to triglutamate derivatives, but this enzyme adds residues to the α-carboxyl rather than the γ-carboxyl of the terminal glutamyl residue (Fig. 5). Conditions governing the expression of this second folylpolyglutamate synthase have not been studied, nor has the gene encoding this protein been identified.

H₂PteGlu₆ and the Assembly of T4 Phage.

Bacteriophage T4 infection of E. coli has been shown to cause alterations in the metabolism of folate derivatives. Infection is associated with an increase in the chain length of folylpolyglutamates and particularly the accumulation of hexaglutamate derivatives (43, 50). H₂PteGlu₆ was shown to be a structural component of the phage baseplate (42), and six molecules are present in the baseplate. Four structural proteins in the phage T4D are the products of genes 29, coding for a protein with folylpolyglutamate synthetase activity, 28, coding for a protein with folylpolyglutamate hydrolase (γ-glutamyl carboxypeptidase) activity (64), td, coding for thymidylate synthase, and frd, coding for dihydrofolate reductase. In cells infected with T4D 28 mutants, there was a marked increase in the amount of very large folylpolyglutamates, and up to 8% of the cell folates contained as many as 12 to 14 glutamate residues (44, 45). The phage-induced folylpolyglutamate synthetase used dihydrofolate derivatives almost as well as tetrahydrofolate derivatives (64), in contrast to FolC, for which H₂PteGlu₁ is a very poor substrate, while the phage-induced hydrolase appeared to degrade long-chain-length dihydropteroylpolyglutamates to the hexaglutamate. H₂PteGlu₆ appears to play a critical role in linking together the six wedge-shaped elements that form the baseplate (41). The pteridine portion of the folate is thought to bind to a site on the phage dihydrofolate reductase, which is a component of the central plug. The long flexible polyglutamate appears to form a flexible bond between the proximal end of the phage long tail fiber and the baseplate.

ACKNOWLEDGMENTS

Work from the Nichols laboratory has been funded by grant AI25106 from the National Institutes of Health. Studies from the Matthews laboratory have been funded by R37 GM24908 from the National Institutes of Health.