Chapter 31

Biosynthesis of Cysteine

NICHOLAS M. KREDICH

INTRODUCTION

The synthesis of l-cysteine from inorganic sulfur is the predominant mechanism by which reduced sulfur is incorporated into organic compounds; it produces significant quantities of l-cysteine only in plants and microorganisms, including Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium) and Escherichia coli. In this process, inorganic sulfate, the most abundant source of utilizable sulfur in the aerobic biosphere, is taken up and reduced to sulfide, which is then incorporated into l-cysteine in a step that is equivalent to the fixation of ammonia into glutamine or glutamate. l-Cysteine is then used for protein synthesis and is converted to glutathione or donates its reduced sulfur to methionine, lipoic acid, thiamin, coenzyme A (CoA), molybdopterin, and other organic molecules.

Sulfate reduction by S. typhimurium and E. coli is assimilatory, i.e., only sufficient sulfur for biosynthetic purposes is reduced, and differs in several ways from dissimilatory sulfate reduction, which is carried out by certain anaerobic microorganisms as part of a respiratory pathway that utilizes sulfate as a terminal electron acceptor and produces large quantities of sulfide as an end product (148). Since sulfate uptake and reduction require a large number of transport and enzyme activities, most of the machinery of cysteine biosynthesis is dedicated to sulfide synthesis. If sulfide is available in the environment, l-cysteine biosynthesis is a relatively simple two-step process requiring conversion of l-serine to O-acetyl-l-serine, which then reacts with sulfide.

Two additional mechanisms for sulfur fixation have been described in S. typhimurium and E. coli. The first occurs through the reaction of thiosulfate with O-acetyl-l-serine to form the thiosulfonate S-sulfocysteine, which is then reduced to l-cysteine (127). This pathway, first described in Aspergillus nidulans (129), is utilized for aerobic growth on thiosulfate and perhaps during anaerobic growth on sulfate (see below). The second mechanism involves the reaction of O-succinyl-l-homoserine with sulfide to form homocysteine in a reaction catalyzed by cystathionine γ-synthase (45, 172). This reaction is probably physiologically insignificant owing to its requirement for very high sulfide concentrations, and even when such concentrations are present, the reaction does not totally satisfy cellular sulfur requirements, because S. typhimurium and E. coli cannot utilize the sulfur moiety of l-homocysteine for l-cysteine biosynthesis. In yeast cells, however, the synthesis of homocysteine from O-succinyl-l-homoserine and sulfide represents either the major or the sole mechanism of sulfur fixation (19).

BIOSYNTHETIC PATHWAY AND GENETICS

Sulfur Utilization

S. typhimurium and E. coli can utilize a variety of inorganic and organic compounds, including sulfate, sulfite, thiosulfate, sulfide, l-cystine, l-cysteine, l-cysteine sulfinic acid, l-djenkolate, lanthionine, and glutathione, as sole sulfur source. Although l-methionine cannot be converted to l-cysteine, it does fulfill about 50% of the total sulfur requirements and will support the growth of some leaky cys mutants. E. coli can also utilize the sulfur moieties of certain alkyl sulfonates, such as cysteic acid, isethionate, and taurine, but S. typhimurium cannot (185, 186). In liquid medium, approximately 70 μM sulfur is required to obtain a density of 10⁹ cells per ml (96). "Sulfur-free" medium E (188), in which MgCl₂ is substituted for MgSO₄, still contains about 7 μM sulfate as a contaminant from other components.

The l-cysteine biosynthetic pathway is shown in Fig. 1, and the genes encoding its activities are listed in Table 1 together with references pertaining to their cloning and sequence analysis. Most cys mutants are defective in one of the steps of sulfate reduction (Fig. 1A) and are in effect sulfide auxotrophs, which can be further distinguished by their responses to forms of reduced inorganic sulfur such as thiosulfate and sulfite (Table 2) (24). cysE mutants cannot synthesize the l-cysteine precursor O-acetyl-l-serine and will not grow on any form of inorganic sulfur unless this product is provided (Fig. 1B). cysB mutants lack a specific transcription activator and are deficient in expression of the entire sulfate reduction pathway, but they do grow on sulfide. Since there are two O-acetylserine (thiol)-lyase isozymes for the reaction of O-acetyl-l-serine and sulfide, mutants lacking only one activity, i.e., cysK or cysM, are still Cys⁺, but cysK cysM strains do not grow on sulfide or on O-acetyl-l-serine. cysM strains cannot utilize thiosulfate because they lack the O-acetylserine (thiol)-lyase isozyme that also catalyzes a reaction between O-acetyl-l-serine and thiosulfate. For unknown reasons, these strains are also cysteine bradytrophs when grown anaerobically on sulfate (43) (see below).

Fig. 1Biosynthesis of L-cysteine in S.typhimurium and E.coli. (A) Sulfate reduction pathway; (B) activation of serine, conversion of serine to cysteine, thiosulfate uptake, and incorporation of thiosulfate into S-sulfocysteine. The existence of a specific thiosulfonate reductase is hypothetical.

Source:

Table 1Genes and activities of the cysteine biosynthetic pathway

Table 2Nutritional characteristics of cysteine auxotrophs

Selection for cys Mutants

Methods for positive selection of cys mutants include chromate resistance for sulfate transport mutants, i.e., cysTWA (143); azaserine resistance for sulfide auxotrophs and cysK mutants and for cysM mutants in a cysK background (64, 65); selection for Leu⁺ in an S. typhimurium leu-500 strain for Δ(topA cysB) mutants (124); and resistance to 1,2,4-triazole for cysK mutants, constitutive cysB mutants, and a promoter-up cysE mutant (62, 63, 66, 176). cysK mutants can be scored by their appearance as white colonies on bismuth ammonium citrate agar (44). Members of one subclass of S. typhimurium 1,2,4-triazole-resistant cysK mutants, originally designated trzB, are unstable and may result from a reversible transposition of cysK from the chromosome to an autogenous plasmid (62).

Cystine and Cysteine Transport

Little is known about l-cysteine transport in enteric bacteria, but its sensitivity to osmotic shock in E. coli implies the participation of a periplasmic binding protein (34). l-Cystine transport in S. typhimurium is mediated by three different uptake systems designated CTS-1, CTS-2, and CTS-3 (5); the genes for these systems have not been identified. CTS-1 is a saturable system with a K_m of 2 μM and a V _max of 9 nmol/min/mg of protein in sulfur-limited cells. Loss of this activity in osmotically shocked cells implies participation of a periplasmic binding protein, which has been characterized from E. coli (11). Its amino acid sequence has been partially determined (16). CTS-1 is regulated as part of the cysteine regulon (see below) and repressed by growth on l-cystine. Constitutive expression of this activity in a cysB(Con) regulatory mutant results in l-cystine sensitivity (5), most likely because of the inhibitory effects of l-cysteine on metabolic processes such as homoserine biosynthesis (33, 51).

CTS-2 is also a saturable system; it has a K_m of 0.1 μM and a V _max of only 0.3 nmol/min/mg of protein. It is not known to be part of the cysteine regulon. CTS-3 is an unsaturable system with a capacity of 0.04 nmol/min/mg of protein per μM l-cystine and probably represents passive diffusion. Mutational loss of CTS-1 or repression by growth on l-cystine does not impair l-cystine utilization under usual laboratory conditions, which employ 0.1 to 1 mM l-cystine, because the activity of CTS-3 alone can satisfy the sulfur requirement of 3.6 nmol of sulfur per min/mg of protein in cells growing with a doubling time of 50 min (96).

E. coli has two kinetically distinct uptake system for l-cystine, of which one is shared with diaminopimelic acid and several l-cystine analogs and the other is more specific (11). Both are associated with periplasmic binding proteins. Although the regulation of these systems has not been studied in detail, the loss of the less specific system by growth on rich medium suggests that this system may be part of the cysteine regulon.

Sulfate and Thiosulfate Transport

Sulfate and thiosulfate uptake in E. coli and S. typhimurium are achieved through a single periplasmic transport system (38, 86) that utilizes two different periplasmic binding proteins for these anions (57). Sulfate uptake by intact cells has a K_m of 36 μM and is inhibited by sulfite, selenate, chromate, and molybdate, which are probably substrates for sulfate-thiosulfate permease (38). Kinetic studies indicate that selenate and selenite share a single transporter with sulfate (108), but molybdate also has a separate transport system (81, 101, 167). In vivo binding of sulfate to the sulfate-binding protein occurs with a K_d of approximately 0.1 μM and is inhibited by chromate but not by thiosulfate or molybdate (142). Thiosulfate-binding protein appears to have little affinity for sulfate (57). Inhibition studies suggest that sulfite can be transported by sulfate-thiosulfate permease, but the abilities of cysTWA mutants to grow on sulfite indicate the existence of some other mechanism as well. Sulfide uptake has not been studied but may occur by diffusion, in a manner similar to that of water uptake.

Components of the sulfate-thiosulfate permease are encoded by the contiguous genes cysP, cysT, cysW, and cysA and by the unlinked gene sbp (52). (The cysT designation has also been used for a cysteinyl-tRNA gene in E. coli [47, 92, 117].) Sulfate uptake in E. coli also requires another gene, cysZ, which is closely linked to cysK (144). An open reading frame that may represent cysZ was found immediately upstream of cysK (17), but deletion of it in S. typhimurium does not impair sulfate utilization (64).

The cysPTWAM cluster from E. coli was cloned, and its entire sequence was reported (57, 173); the same region from S. typhimurium was cloned (64, 122), but only the cysP and cysM sequences were reported (57, 175). By analogy with other periplasmic transport systems (1), cysT and cysW encode homologous peptides that probably span the membrane and form a channel for the passage of sulfate, thiosulfate, and related anions (173). cysA encodes a third component, presumed to be membrane associated, that is homologous to the nucleotide-binding peptides characteristic of this class of transport systems. These three genes probably represent the three complementation groups formerly recognized as cysA_a, cysA_b, and cysA_c in S. typhimurium (121).

sbp and cysP encode the sulfate and thiosulfate periplasmic binding proteins, respectively (52, 57). S. typhimurium sulfate-binding protein is a 32-kDa monomer that has been purified, had its sequence determined, and been characterized by X-ray crystallography (74, 142, 149). The sequence deduced from E. coli sbp predicts a 19-amino-acid hydrophobic signal peptide (52). Thiosulfate-binding protein sequences deduced from the E. coli and S. typhimurium cysP genes are 45% identical to Sbp and include 25-residue signal peptides. The failure to find sbp mutants among a large collection of S. typhimurium sulfate permease mutants (132) suggests that Sbp is not absolutely required for sulfate uptake under laboratory conditions, where concentrations are relatively high (∼1 mM). Likewise, mutational loss of thiosulfate-binding protein in E. coli does not cause cysteine auxotrophy, but it does result in a 50% decrease in growth rate at 0.02 mM thiosulfate and a sevenfold reduction in the rate of uptake at 0.01 mM thiosulfate (57).

Sulfate Activation

The reduction of sulfate requires its prior activation to a phosphosulfate mixed anhydride. This activation is achieved by the ATP sulfurylase-catalyzed reaction of sulfate with ATP to give adenosine 5'-phosphosulfate (APS) and PP_i (155). A second enzyme, APS kinase, phosphorylates APS with another ATP to give PAPS (154), which is then enzymatically reduced by PAPS sulfotransferase (also known as PAPS reductase) to sulfite. ATP sulfurylase is encoded by cysD and cysN, and APS kinase is encoded by cysC. The three genes are contiguous in the cysDNC cluster, which was cloned from E. coli and has had its entire sequence determined (106, 107). This region was cloned in S. typhimurium (122) but not sequenced.

The extremely low equilibrium constant (10^–8) of the reversible ATP sulfurylase reaction in the forward direction (155) has for years led to the assumption that sulfate activation must be driven through the efficient hydrolysis of PP_i combined with the conversion of APS to PAPS. Recently, an additional factor has come to light through the discovery that the E. coli enzyme contains two nonidentical subunits, a 35-kDa catalytic subunit encoded by cysD and a 53-kDa peptide encoded by cysN that has a deduced sequence homologous to those of GTP-binding proteins such as E. coli EF-Tu and RAS (106, 107). The native enzyme is thought to contain four each of the two different subunits (109). GTP stimulates the rate of APS synthesis as much as 116-fold with an apparent K_m of 19 μM and is hydrolyzed in the process (105). If coupled to APS synthesis, GTP hydrolysis would lower the apparent K _eq for the overall reaction by a factor of about 10⁵ (104). The enzyme mechanism appears to involve the GTP-dependent formation of an AMP∼enzyme reaction intermediate, which then undergoes nucleophilic attack by sulfate to give APS (109).

E. coli APS kinase has been purified to homogeneity (161, 165) and is composed of identical 22-kDa peptides encoded by cysC (106, 107, 160). The peptide subunit is phosphorylated by ATP at Ser-109 (160) and exists as a dimer in the phosphorylated state and mostly as a tetramer in the unphosphorylated state (161). The phosphorylated enzyme probably represents an intermediate on the reaction pathway and can donate its phosphate either to APS to give the product PAPS or to ADP to re-form the substrate ATP.

PAPS Reduction

Although many organisms synthesize PAPS as a donor for organic sulfate ester formation (a process not known to occur in S. typhimurium and E. coli), reduction of this compound is confined to plants and microorganisms (31, 37). E. coli PAPS sulfotransferase is a homodimer of 28-kDa subunits (100, 181) that are encoded by cysH in the cysJIH operon (39, 99, 140). Thioredoxin is believed to serve as the physiologic reductant, but trxA mutants, which lack this factor, are Cys⁺ because they can substitute another reductant, glutaredoxin, which is encoded by grx (180, 183). trxA grx strains are Cys^– and accumulate secondary mutations that prevent sulfate uptake or activation of sulfate to PAPS unless they are maintained on sufficient cysteine to repress these activities (156). A similar phenomenon has been observed in aged cultures of cysH mutants (49) and in cysQ mutants (131) and is probably due to PAPS toxicity. cysQ is postulated to encode a protein involved in PAPS metabolism.

The enzyme mechanism for PAPS sulfotransferase is thought to involve transfer of a sulfo moiety from PAPS to one of the two redox sulfhydryl groups of thioredoxin to give an organic thiosulfate, thioredoxin-S-SO₃ ^– (164, 182). An enzyme-S-SO₃ ^– intermediate that transfers the sulfo group to thioredoxin (100) has been proposed, and the deduced amino acid sequences of the S. typhimurium and E. coli PAPS sulfotransferase show a single cysteine residue, which would have to be the acceptor site in such a mechanism (100, 140). Once formed, thioredoxin-S-SO₃ ^– rearranges to give free sulfite and oxidized thioredoxin, which is regenerated by thioredoxin reductase. Thioredoxin-S-SO₃ ^– may also be reduced to a hydrodisulfide (R-S-SH) before it donates a sulfide moiety to O-acetyl-l-serine (182), but such a mechanism would bypass NADPH-sulfite reductase, and it is clear that E. coli and S. typhimurium mutants lacking this enzyme do not grow on sulfate.

Sulfite Reductase

During aerobic growth, the reduction of sulfite to sulfide is catalyzed by NADPH-sulfite reductase (171), and S. typhimurium mutants lacking this enzyme accumulate sulfite from sulfate, implying that sulfite is a normal intermediate in assimilatory sulfate reduction (39). NADPH-sulfite reductase also exhibits nitrite reductase activity, which is of doubtful physiologic significance. Under anaerobic conditions, S. typhimurium expresses another type of sulfite reductase, which is discussed below.

NADPH-sulfite reductase contains nonidentical subunits arranged with a stoichiometry of α ₈ β ₄, where α is a 66-kDa flavoprotein encoded by cysJ, and β is a 64-kDa hemoprotein encoded by cysI in the cysJIH operon (168). Both genes have been cloned from E. coli and S. typhimurium, and their sequences have been determined (133, 140). The role of the flavoprotein is to accept electrons from NADPH and transfer them to the hemoprotein, which then reduces sulfite. Although α and β subunits are tightly associated in the holoenzyme, purified preparations of each have been obtained from the E. coli enzyme by dissociation in urea (168) or from S. typhimurium by purifying one component from a mutant unable to express the other (170).

The free flavoprotein exists as an octamer that contains both flavin dinucleotide (FAD) and flavin mononucleotide (FMN) and has substantial NADPH-cytochrome c reductase and other diaphoraselike activities (168). The flavoprotein transfers electrons in the sequence NADPH → FAD → FMN → hemoprotein (169). The deduced sequence is homologous to that of rat liver cytochrome P-450 reductase (133, 150), which also contains FAD and FMN (76). The FAD-binding domains of both proteins appear to have been derived from the same ancestral gene as ferredoxin-NADPH reductase and NADH cytochrome c ₅ reductase, whereas the FMN-binding domain is homologous to that of bacterial flavodoxins. Whereas cytochrome P-450 reductase exists as a 77-kDa monomer that contains one molecule each of these two cofactors, the octameric sulfite reductase flavoprotein contains only four FADs and four FMNs, implying that the subunits are arranged in such a manner that binding of one flavin precludes binding of a second to the same subunit.

The free sulfite reductase hemoprotein is a monomer and contains one Fe₄S₄ cluster and one siroheme, an unusual heme found thus far only in sulfite and nitrite reductases (125, 126, 168, 187). The sequence of electron flow is flavoprotein → Fe₄S₄ cluster → siroheme → sulfite, and reduced methylviologen can be substituted for the flavoprotein and NADPH. The deduced E. coli and S. typhimurium sequences include four cysteine residues arranged in two clusters as Ala-Cys-X₅-Cys separated by 37 residues from Gly-Cys-Pro-Asn-Gly-Cys (140). X-ray crystallographic studies (119) showed that these four cysteines bind the Fe₄S₄ cluster, and spectroscopic studies indicate that one of these serves as a bridging ligand, which electronically couples the cluster to siroheme (20, 23, 87, 88). This arrangement of cysteines may be regarded as a "siroheme motif" and has been noted with variations in other siroheme enzymes, including an assimilatory sulfite reductase from Desulfovibrio vulgaris (178); the anaerobically expressed sulfite reductase from S. typhimurium (61); nitrite reductases of bacteria, fungi, and spinach (4, 90, 147); and a dissimilatory sulfite reductase from an extremely thermophilic archaeal species, Archaeoglobus fulgidus (32).

Synthesis of the siroheme moiety of NADPH-sulfite reductase requires an S-adenosylmethionine-dependent uroporphyrinogen III methylase encoded by cysG (189), which is also required for cobalamin synthesis. Thus, cysG mutants are deficient in three functions: sulfite reductase, nitrite reductase, and cobalamin synthesis (25, 80). The cysG sequences from E. coli and S. typhimurium have been determined. The gene is contiguous with nir genes involved in NADPH-nitrite reductase expression (147, 193). Siroheme synthesis is a limiting factor for overexpression of NADPH-sulfite reductase from a plasmid, but this deficiency can be overcome by including a copy of cysG on the expression vector (193).

Serine Transacetylase and Cysteine Synthase

Serine transacetylase catalyzes the acetylation of l-serine by acetyl-CoA to give O-acetyl-l-serine, the direct precursor of l-cysteine (95, 98). The enzyme is feedback inhibited by l-cysteine, thus providing kinetic regulation of this short branch of the pathway. Furthermore, mutant strains lacking serine transacetylase are defective in expression of the enzymes of sulfate reduction because O-acetyl-l-serine is the precursor of N-acetyl-l-serine, the inducer of the cysteine regulon (see below).

Serine transacetylase resides in a multifunctional complex termed cysteine synthase, which also contains O-acetylserine (thiol)-lyase-A, one of two enzymes catalyzing the synthesis of l-cysteine from O-acetyl-l-serine and sulfide (8, 95). Serine transacetylase and O-acetylserine (thiol)-lyase-A are encoded by cysE and cysK, respectively (44, 67, 82, 98), and have predicted subunit masses of 30 and 35 kDa, respectively (17, 36, 103, 175). The complex has a mass of approximately 309 kDa but readily forms aggregates that are two and four times that size (30, 95). The smallest form contains four O-acetylserine (thiol)-lyase-A subunits and four to six serine transacetylase subunits; the exact number is not known.

Ordinarily, serine transacetylase is much less abundant than O-acetylserine (thiol)-lyase-A and is isolated as the cysteine synthetase complex (95). The free enzyme is found, however, in mutants that overproduce serine transacetylase (66, 102) or lack O-acetylserine (thiol)-lyase-A (67). The Cys⁺ phenotype of the latter class of mutants indicates that the complex is not required for in vivo function of serine transacetylase. In vitro, O-acetyl-l-serine dissociates the cysteine synthetase complex into a serine transacetylase multimer with a mass of about 160 kDa and two 70-kDa O-acetylserine (thiol)-lyase-A dimers, which can be resolved from each other by chromatography and then reassociated into the cysteine synthetase complex (95). The dissociation reaction has a K _0.5 for O-acetyl-l-serine of about 20 μM and is inhibited by sulfide. The physiologic role of the dissociation reaction is not known, but it is interesting that serine transacetylase may be physically associated with O-acetylserine (thiol)-lyase in plants as well (40). In experiments designed to determine whether the cysteine synthase complex releases O-acetyl-l-serine prior to its reaction with sulfide, Cook and Wedding (29) found only a slight decrease in the predicted lag time between O-acetyl-l-serine synthesis and reaction, which was consistent with release of this intermediate from the complex but also suggestive of some small kinetic advantage from increased local concentrations of O-acetyl-l-serine.

The kinetic mechanism for the free enzyme is bi-bi ping-pong, with acetyl-CoA being added first with the release of CoA and then l-serine being added with the release of O-acetyl-l-serine (102). Kinetic studies of the cysteine synthase enzyme are complicated by the formation of aggregates with different affinities and various degrees of positive cooperativity for acetyl-CoA (30). The substrate l-serine and the feedback inhibitor l-cysteine alter apparent K_ms for acetyl-CoA through effects on aggregation. At 0.1 mM acetyl-CoA, l-cysteine inhibits with an apparent K_i of about 10^–6 M (95, 98). A mutant enzyme that is at least 10-fold less sensitive to l-cysteine inhibition owing to the substitution of an isoleucine for the methionine at residue 256 has been described (36).

O -Acetylserine (Thiol)-Lyase and S-Sulfocysteine Synthase

Synthesis of l-cysteine from O-acetyl-l-serine and sulfide is catalyzed by two distinct O-acetylserine (thiol)-lyase isozymes designated -A and -B and encoded by cysK (17, 44, 67, 103) and cysM (65, 173, 174, 175). The deduced amino acid sequences of the two E. coli isozymes are 43% identical. Mutational loss of either isozyme does not appreciably affect aerobic growth on sulfate, but the -A isozyme probably plays the larger role because its activity is at least 10-fold higher under these conditions (65). O-Acetylserine (thiol)-lyase-B is more important for growth on thiosulfate and for anaerobic growth on sulfate (see below).

O-Acetylserine (thiol)-lyase-A is a dimer of 35-kDa subunits that exists both in the free form and complexed with serine transacetylase as cysteine synthase (93, 95). Preliminary data on the X-ray crystallographic structure of the free form have been reported (153). O-Acetylserine (thiol)-lyase-A exhibits wide substrate specificity and in lieu of sulfide can use several different nucleophiles, including 1,2,4-triazole (96), methyl mercaptan (8), 5-thio(2-nitrobenzoate) (177), cyanide (18), and azide (21, 141). Enzymatically active analogs of O-acetyl-l-serine include O-propionyl-l-serine, O-butyryl-l-serine, β-chloro-l-alanine (28), and azaserine (diazoacetyl-l-serine) (65). Reactivity with azaserine releases the toxic product diazoacetic acid, thus providing a method of positive selection for mutants deficient in either O-acetylserine (thiol)-lyase or sulfide synthesis (see above).

O-Acetylserine (thiol)-lyase-A contains one pyridoxal phosphate per subunit, and this moiety forms a Shiff base with the lysine residue at position 42 (130). The overall kinetic mechanism is bi-bi ping-pong, with O-acetyl-l-serine binding first and displacing Lys-42 to form its own Shiff base with pyridoxal phosphate (27, 28, 177). Acetate is then released by β elimination, leaving a pyridoxal phosphate-bound α-aminoacrylate intermediate that undergoes nucleophilic attack by sulfide to form l-cysteine. K_ms for the free enzyme are 1.0 mM for O-acetyl-l-serine and 6 μM for sulfide (177). The enzyme is product inhibited by l-cysteine but only at concentrations that are too high (∼3 mM) to be of physiologic significance. Formation of the cysteine synthase complex alters certain kinetic properties of O-acetylserine (thiol)-lyase-A, decreasing the V _max by 50% and increasing the apparent K_m for O-acetyl-l-serine about fourfold (95).

O-Acetylserine (thiol)-lyase B is also a dimer of identical subunits, which have a predicted mass of 33 kDa each (127), but is not known to form a complex with serine transacetylase (9, 65). It contains pyridoxal phosphate and has a kinetic mechanism that is identical to that of the -A isozyme. K_ms are 0.9 mM for O-acetyl-l-serine and 10 μM for sulfide, and the V _max is approximately 90% that of O-acetylserine (thiol)-lyase-A (177). O-Acetylserine (thiol)-lyase-B differs from the -A isozyme by its ability to use thiosulfate as a nucleophile, giving S-sulfocysteine (R-S-SO₃) as a product (127). The apparent K_m for thiosulfate in this reaction is 2.7 mM. The conversion of S-sulfocysteine to cysteine has not been characterized in S. typhimurium and E. coli but could involve hydrolysis to cysteine and sulfate or reduction by glutathione to cysteine and sulfite (192). The advantage of this relatively short pathway is that it obviates the need for sulfate reduction by directly incorporating the sulfane moiety of thiosulfate into an organic form that requires only a one- or two-electron reduction (depending on whether sulfate or sulfite are by-products) to form cysteine. The physiologic importance of this activity is questionable during aerobic growth on sulfate, since cysM mutants grow quite well, and thiosulfate is not recognized as an intermediate in sulfate assimilation by S. typhimurium and E. coli. cysM mutants do lack the ability to utilize thiosulfate aerobically (128, 174), however, and are cysteine bradytrophs during anaerobic growth on sulfate, suggesting that thiosulfate may be an intermediate of sulfate reduction under these conditions (43).

REGULATION

l-Cysteine biosynthesis in S. typhimurium and E. coli ceases almost entirely when cells are grown on l-cysteine or l-cystine, owing to a combination of end product inhibition of serine transacetylase by l-cysteine and a gene regulatory system known as the cysteine regulon, wherein genes for sulfate assimilation are expressed only when reduced sulfur is limiting. Enzyme degradation may play a regulatory role as well. The first two mechanisms are interdependent, since the inducer for transcription activation, N-acetyl-l-serine, is derived from the serine transacetylase product, O-acetyl-l-serine. This relationship provides communication between branches of the pathway, allowing l-cysteine to regulate genes for sulfate assimilation and thiosulfate uptake negatively by inhibiting serine transacetylase (Fig. 2). Sulfur limitation derepresses these genes by relieving end product inhibition of serine transacetylase with a resultant accumulation of O-acetyl-l-serine and N-acetyl-l-serine.

Fig. 2Interrelationships of control mechanisms for L-cysteine biosynthesis. L-Cysteine feedback inhibits synthesis of O-acetyl-L-serine, which is the precursor for the inducer N-acetyl-L-serine. Sulfide and thiosulfate also exert end product inhibition in their capacities as anti-inducers and by their abilities to react with and deplete O-acetyl-L-serine.

Source:

A second type of end product regulation results from the ability of sulfide and thiosulfate to act as anti-inducers, with effects on the cysteine regulon that are equivalent to the inhibition of inducer synthesis by l-cysteine. Thus, l-cysteine, sulfide, and thiosulfate are all negative regulators of l-cysteine biosynthesis (Fig. 2). Other sulfur compounds, such as sulfite and methionine, can partially downregulate the system but do so indirectly through their abilities to serve as readily utilized sulfur sources and to increase intracellular levels of sulfide and l-cysteine (190).

Kinetic Regulation

End product inhibition of serine transacetylase by l-cysteine represents the major, and probably the only, physiologically significant form of kinetic regulation in the pathway. The efficacy of this mechanism is illustrated by the fact that a sixfold increase in levels of wild-type serine transacetylase does not affect expression of the cysteine regulon (66), but a mutant with a normal level of a feedback-resistant enzyme excretes l-cysteine up to a concentration of about 2.5 mM (36), which is about 36 times the amount required for growth to a cell density of 10⁹ cells per ml (96).

Both O-acetylserine (thiol)-lyase isozymes are inhibited by l-cysteine and sulfide but at concentrations that are too high to be of regulatory importance (177). In vitro studies have uncovered no evidence of significant kinetic inhibition within the sulfate reduction pathway or in thiosulfate uptake (38, 41), and a regulatory mutant that cannot repress the sulfate reduction pathway excretes large amounts of sulfide, implying the lack of kinetic inhibition in vivo (14). The inability to feedback inhibit sulfate uptake and reduction poses a potential problem of synthesizing excess sulfide when a sudden shift from sulfur limitation to sulfur abundance occurs. It may be significant in this regard that ATP sulfurylase and APS kinase activities rapidly decay in cells depleted for O-acetyl-l-serine, thereby shutting off the pathway at an early step (96). This mechanism may be particularly useful in preventing toxicity from PAPS accumulation (49, 131, 156).

Overview of the Cysteine Regulon

The cysteine regulon comprises those genes participating in l-cysteine synthesis and l-cystine transport that either are regulated by sulfur availability or participate in this response (reviewed in reference 94). This regulon includes the genes for l-cystine, sulfate, and thiosulfate uptake; sulfate activation and reduction to sulfide; and both O-acetylserine (thiol)-lyase isozymes (Table 1). All of these genes are positively regulated by the transcription activator CysB and the inducer N-acetyl-l-serine. cysG is an exception, and although required for sulfite reduction, it is not part of the cysteine regulon (193).

cysB and cysE are also considered part of the cysteine regulon because they encode the specific transcription activator CysB and serine transacetylase, which synthesizes the immediate precursor of N-acetyl-l-serine. cysB itself is negatively autoregulated (12, 77, 139), but cysE expression may not be regulated at all. cysB and cysE mutants are pleiotropic and are not derepressed for activities of the cysteine operon by sulfur limitation (82, 83, 93), but this defect can be overcome in cysE strains by either O-acetyl-l-serine or N-acetyl-l-serine (137). O-Acetyl-l-serine also supports the growth of cysE mutants on inorganic sulfur, but N-acetyl-l-serine cannot do so, because it is not converted to O-acetyl-l-serine, which is required as a substrate for l-cysteine biosynthesis.

Sulfur limitation is a necessary condition for depression of the cysteine regulon (85, 93) not only because l-cysteine inhibits serine transacetylase and inducer synthesis but also because sulfide and thiosulfate are anti-inducers (58, 138). Sulfide prevents derepression of the pathway by exogenous O-acetyl-l-serine and N-acetyl-l-serine (138), and l-cysteine (or l-cystine) does the same because it is degraded to sulfide by the inducible enzyme cysteine desulfhydrase (97). Maximal derepression occurs during growth on the limiting sulfur sources glutathione and l-djenkolic acid (39); sulfate, sulfite, and thiosulfate give intermediate levels of derepression; and sulfide, l-cystine, and l-cysteine provide maximum repression (93, 145, 191). In E. coli, this order of sulfur sources correlates directly with intracellular l-cysteine and inversely with ATP sulfurylase and APS kinase levels (190). Wild-type and even cysB strains grow well on sulfide because repressed levels of the two O-acetylserine (thiol)-lyase isozymes are still sufficient for an adequate rate of l-cysteine biosynthesis (93).

Transcription Units

A total of 22 cys genes have been designated, and this total does not include those for the l-cystine transport system CTS-1 (Table 1). Most encode activities directly required for cysteine biosynthesis, while others function indirectly in l-cysteine and inorganic sulfur metabolism and are not known to be part of the cysteine regulon. The latter group includes cysL, the designation for a class of Cys⁺ selenate-resistant mutants with lesions in or near cysPTWAM that may have an altered sulfate permease that is selectively defective in selenate uptake(62); cysQ, which encodes a protein that protects against PAPS toxicity (131); cysS, the gene for cysteinyl-tRNA ligase (13, 42, 55); an E. coli cysteinyl-tRNA gene designated cysT (47, 92), which is distinct from the sulfate permease gene cysT (173); cysX, an open reading frame of unknown significance that overlaps E. coli cysE (179); cysZ, which is required for sulfate transport in E. coli but not in S. typhimurium (17, 144); and cysG, which is required for siroheme synthesis but is regulated from the nirB promoter as part of a cluster of genes involved in the anaerobic expression of nitrite reductase (114, 146, 147). Constitutive expression of cysG provides sufficient siroheme for sulfate assimilation. The remaining 15 known genes are known members of the cysteine regulon and are found at five locations on the chromosome in at least seven different transcription units (Table 3). CTS-1 genes are also regulated as part of the cysteine regulon (5) and must compose an eighth transcription unit.

Table 3Transcription units of the cysteine regulon

cysPTWAMK Region.

Translational coupling of genes in the cysPTWA cluster (173) indicates that these genes are expressed as a single transcription unit from a promoter just upstream of cysP (57, 58). cysM is separated from cysA by only 174 bp in E. coli and may also be part of this operon, which is transcribed counterclockwise on the chromosome. cysK is separated from cysPTWAM by about 7 kb and is transcribed clockwise from its own promoter (17, 123). An open reading frame, which may correspond to E. coli cysZ (144), is located just upstream of cysK, but nothing is known about its product or regulation (17).

cysCND-cysHIJ Region.

The cysDNC cluster comprises a single operon with a promoter immediately preceding cysD (106, 107, 115). The nearby cysJIH cluster is a single operon with a promoter just upstream of cysJ (111, 112, 134, 137) and is separated from cysDNC by approximately 25 kb of DNA in S. typhimurium (75) and at least 11 kb in E. coli (68, 73). The gene order is thyA- -cysJ cysI cysH iap (∼11 kb) cysD cysN cysC in E. coli and thyA- -cysC cysN cysD (∼25 kb) cysH cysI cysJ in S. typhimurium (35, 84, 106, 107, 133, 140), indicating that the cysCND−cysHIJ segment is inverted with respect to other chromosomal loci.

sbp , cysE, and cysB Regions.

sbp, the E. coli gene for sulfate-binding protein, is regulated as part of the cysteine regulon (132) and is found in the gene order pfkA sbp cdh-tpi, where sbp is transcribed clockwise (52). cysE is found in the order xyl mtl cysE rfa-pyrE in both S. typhimurium and E. coli (3, 36, 158, 175) and is transcribed counterclockwise (66). cysE is not regulated as part of the cysteine regulon (93). cysB is found in the order purB- -pyrF- -cysB topA trpA- -aroD in S. typhimurium and purB- -trpA-topA cysB pyrF- -aroD in E. coli, in which the chromosomal region between 25 and 35 min is inverted (70). Transcription is counterclockwise in S. typhimurium and clockwise in E. coli.

Molecular Aspects of the Cysteine Regulon

Transcription start sites for the cysJIH, cysK, cysPTWAM, and cysDNC operons have been determined and define –10 regions, which conform to the consensus TATAAT (17, 57, 58, 120, 137). As observed with many other positively regulated promoters (54, 151), the -35 regions show little or no resemblance to the consensus TTGACA. In vitro studies with the cysJIH, cysK, and cysP promoters have confirmed that these promoters are very inefficient at forming transcription initiation complexes unless CysB and N-acetyl-l-serine are present.

CysB Protein, Inducer, and Anti-Inducers.

CysB is a homotetramer of 36-kDa subunits encoded by cysB (120, 136) and belongs to the LysR family of transcriptional activators (53, 162) with a putative helix-turn-helix DNA binding motif (15) at amino acid residues 19 to 38. Highly purified S. typhimurium CysB has been characterized by in vitro binding, footprinting, and transcription studies. A 233-residue amino-terminal fragment of Klebsiella aerogenes cysB (113) was crystallized and studied by X-ray diffraction (184), but a structure is not yet available.

In vitro experiments showed that CysB binds as a tetramer (59) just upstream of the –35 regions of the cysP, cysJIH, and cysK promoters and activates transcription in the presence of N-acetyl-l-serine (58, 123, 137). It also binds to the +1 region of the cysB promoter, where it acts as a repressor (139). Binding to these promoters occurs in the absence of inducer and is qualitatively and quantitatively altered by N-acetyl-l-serine in ways that vary from one cys promoter to another (see below).

Originally, O-acetyl-l-serine was thought to be the inducer of the cysteine regulon (85, 93), but more recent in vitro and in vivo studies indicate that the true inducer is N-acetyl-l-serine (137). Fluorescence emission spectroscopic studies have shown that N-acetyl-l-serine, but not O-acetyl-l-serine, binds to the closely related K. aerogenes CysB protein with a dissociation constant of about 4 mM and a stoichiometry of 1 molecule per protein subunit (113). It now seems likely that the activity of O-acetyl-l-serine is due to its conversion to N-acetyl-l-serine by a nonenzymatic, intramolecular O- to N- acetyl migration. It is not known whether an enzyme catalyzes this reaction in vivo, nor is anything known about N-acetyl-l-serine degradation.

Effects of N-acetyl-l-serine on in vitro DNA binding and transcription initiation are noted at concentrations of 0.1 to 5 mM (58, 123, 137, 138). The anti-inducers sulfide and thiosulfate reverse these effects at concentrations ranging from 0.5 to 2 mM for sulfide and from 0.025 to 0.25 mM for thiosulfate but do not affect the DNA binding that occurs in the absence of N-acetyl-l-serine (58, 138). Sulfide and thiosulfate do not affect N-acetyl-l-serine binding to K. aerogenes CysB (113), suggesting that these anti-inducers act by preventing or altering conformational changes caused by bound inducer.

CysB Binding Sites.

Nine different binding sites have been identified in four different cys promoters (Fig. 3). Each of the three positively regulated promoters has multiple binding sites, including an activation site located just upstream of the –35 region that is required for transcription activation by CysB (58, 123, 137, 138). These activation sites are designated CBS-J1, CBS-K1, and CBS-P1 for the cysJIH, cysK, and cysP promoters, respectively. N-Acetyl-l-serine stimulates binding to activation sites. The cysB promoter has a single site, a repressor site designated CBS-B, that is centered at the transcription start site and binds with lower affinity in the presence of N-acetyl-l-serine (60, 139). Accessory sites are found both upstream and downstream of activation sites and include CBS-J2 and CBS-J3 in the cysJIH promoter, CBS-K2 in the cysK promoter, and CBS-P2 and CBS-P3 in the cysP promoter (Fig. 3). These sites are of unknown function but may serve to sequester CysB at cys promoters even when sulfur is replete and gene activation is not required. N-Acetyl-l-serine stimulates binding to CBS-J2 and CBS-P2 but inhibits binding to CBS-J3, CBS-K2, and CBS-P3 (58, 60, 123).

Fig. 3Binding sites for CysB in the positively regulated cysJIH,cysK, and cysP promoters and in the negatively regulated cysB promoter. CBS-J1, CBS-K1, and CBS-P1 are activation sites and are required for transcription activation by CysB. CBS-B is a repressor site for cysB negative autoregulation, and the other sites are termed accessory sites. The inducer N-acetyl-L-serine stimulates CysB binding to the shaded sites and inhibits binding to the other sites. The upstream portion of CBS-J3 has not been characterized. Arrows indicate transcription start sites and promoter ?35 regions.

Source:

Hydroxyl-radical footprints and nucleotide sequence alignments indicate that CysB binding sites are composed of 19-bp half-sites that vary in their spacing and relative orientations in patterns that can be correlated with the effects of N-acetyl-l-serine on CysB binding (60). Activation sites are composed of a pair of convergently oriented half-sites, designated a and b, that are separated by 1 bp in CBS-J1 and CBS-P1 and by 2 bp in CBS-K1 (Fig. 4). This spacing allows CysB to bind to one side of the DNA duplex over a span of about 40 bp. The same topology is found in CBS-J2 and CBS-P2, except that the half-site separation in CBS-P2 is 3 bp. The increased binding affinities of all five sites in the presence of N-acetyl-l-serine are thought to be a consequence of this particular half-site arrangement.

Fig. 4Correlation between half-site organization, CysB- induced DNA bending, and effects of N-acetyl-L-serine. Half-sites are designated a, b, and in one case c and are oriented as indicated. Half-site pairs, i.e., binding sites, with increased affinities in the presence of N-acetyl-L-serine are shaded. Bend points induced by CysB binding that are sensitive to N-acetyl-L-serine are indicated on the cysK and cysP promoters by vertical arrows.

Source:

Half-site arrangements in CBS-B and CBS-K2 are similar to each other but quite different from those of activation sites (60). Here, half-sites are divergently oriented and separated by one or two helical turns, i.e., 21 bp in CBS-B and 11 or 23 bp in CBS-K2. This arrangement is thought to result in decreased binding affinity in the presence of N-acetyl-l-serine. As with activation sites, CysB binds to a single helical face in CBS-B and CBS-K2, but here it bends the DNA to give an overall footprint of approximately 60 bp (see below). The CBS-K2 region actually contains three half-sites, designated K2a, K2b, and Kc (Fig. 4). Without N-acetyl-l-serine, binding is to the upstream CBS-K2a half-site and the closer of two downstream half-sites, CBS-K2c, whereas in the presence of inducer, binding shifts from CBS-K2c to the more distant downstream half-site CBS-K2b.

Binding to CBS-P3 is also inhibited by N-acetyl-l-serine (58), but in this case the effect is due to an upstream lateral movement of CysB from CBS-P3 to the overlapping CBS-P1 site. CBS-P3 is actually a combination of the downstream half-site of CBS-P1 and an extra half-site located between CBS-P1 and CBS-P2. This topology is thought to result in DNA bending that is responsive to N-acetyl-l-serine (see below).

CysB-Induced DNA Bending.

In the absence of N-acetyl-l-serine, CysB induces a 100° bend just upstream of CBS-K1 in the cysK promoter by binding of a single tetramer to the activation site and the CBS-K2c half-site (59, 123). This interaction is thought to enlist three different CysB subunits, each binding to a different half-site, and is prohibited by N-acetyl-l-serine, which reduces the bend angle to about 50° by causing CysB to bind exclusively to CBS-K1. A similar phenomenon has been noted for the cysP promoter: CysB binds to CBS-P1 and CBS-P3b in the absence of N-acetyl-l-serine (58, 59, 60). This binding induces a 100° bend just downstream of CBS-P1, and N-acetyl-l-serine reduces the bend by causing CysB to bind only to CBS-P1. For both promoters, the arrangement of a half-site oriented toward an activation site but separated from it by a single helical turn results in induced DNA bending that is sensitive to N-acetyl-l-serine. The significance of such bending to transcription regulation is questionable, because mutant promoters lacking CBS-K2 are not bent and appear to function normally in vitro and in vivo (123). Bending of CBS-K2 and CBS-B (see above) involves different half-site combinations and is not sensitive to N-acetyl-l-serine.

Mechanism of Transcription Activation by CysB.

CysB is thought to employ a helix-turn-helix motif for DNA binding, and two mutations affecting this putative region, one in E. coli and the other in S. typhimurium (26), result in a loss of autoregulation, implying that they cannot bind to the cysB promoter region. Presumably, they are defective in binding to activation sites as well.

Amino acid substitutions in LysR family proteins that allow transcription activation in the absence of inducer, i.e., a constitutive phenotype, are generally located in the center of the polypeptide, implying that this region is important in effector recognition and response (7, 118, 163). Constitutive cysB mutants are relatively easy to isolate because they cause resistance to 1,2,4-triazole (26, 56, 176). Residue T149 appears to be very important in determining the response to inducer, and 9 of the 19 possible substitutions at this position are partially or totally constitutive for cysK and cysJIH expression during growth on l-cystine (26). Two highly purified mutant proteins, T149M and T149P, activate transcription from the cysK, cysJIH, and cysP promoters in the absence of N-acetyl-l-serine and are insensitive to anti-inducers (T. E. Colyer, Ph.D. thesis, Duke University, Durham, N.C., 1993).

Insertions of as many as 14 amino acids can also be tolerated at Thr-149, and two such proteins are partially constitutive in vivo, indicating that this region probably lies on the surface of the molecule. CysB residue Thr-149 may function like residue 138 in the E. coli catabolite activator protein, which lies in a hinge region involved in a cyclic-AMP-induced allosteric change that increases binding affinity for specific promoter sites and is necessary for transcription activation (89, 157).

The positions of activation sites in the cysK, cysJIH, and cysP promoters suggest that CysB functions as a class I transcription activator (71, 72) and imply that the protein makes contact with the carboxyl-terminal portion of the RNA polymerase α subunit. Such transcription factors are distinguished by the fact that they are inactive with RNA polymerase containing α subunits with deletions or certain point mutations in the C-terminal one-third (69, 194), and two strains with such mutations, rpoA341 in E. coli (48) and rpoA155 in S. typhimurium (110), are Cys^–. Additional evidence for a role for the α subunit lies in the findings that cysB is required for efficient expression of adi, the gene for arginine decarboxylase, and that this process is defective in the E. coli rpoA strain (10).

NADPH-sulfite reductase and O-acetylserine (thiol)-lyase levels in S. typhimurium are lowered by the DNA gyrase inhibitors nalidixic acid and novobiocin, suggesting that expression of these activities is sensitive to the state of DNA superhelicity (135). It is not known whether this effect involves cysB expression or the interaction of CysB with the cysK and cysJIH promoters. A number of prokaryote promoters, including those that are dependent on catabolite repressor protein, are known to be sensitive to DNA superhelicity (159), but there is no evidence to suggest that catabolite repressor protein is involved in expression of the cysteine regulon.

The Cysteine Regulon and Global Control

Given the role of reduced organic sulfur in many different biologic processes, the cysteine regulon can be considered a global control system that might be expected to interact with other metabolic processes. One example of such interaction is the apparent requirement for CysB in the efficient induction of the E. coli adi gene (10), which is discussed above; another is the finding that cysB and cysB mutants are more resistant than wild type to novobiocin (152). In addition, a functional cysPTWA operon is required for the anaerobic induction of aidB, a component of the adaptive response to alkylation damage (116).

ANAEROBIC CYSTEINE BIOSYNTHESIS

Mechanisms for sulfate uptake, activation, and reduction to sulfite and a requirement for O-acetyl-l-serine appear to be the same for aerobic and anaerobic growth in S. typhimurium (6), but cysJ and cysI mutants are Cys⁺ during anaerobic growth owing to the activity of a sulfite reductase that is expressed from the asrABC operon only under anaerobic conditions (50, 61). asrC encodes a peptide that is homologous to the cysI-encoded NADPH-sulfite reductase and has a siroheme-binding motif, asrA encodes a peptide with a ferredoxinlike arrangement of cysteine residues, and asrB may encode a flavoprotein analogous to that of the flavoprotein of the aerobic NADPH-sulfite reductase. A partially purified preparation of this activity preferred NADH over NADPH as a reductant and was reversibly inhibited by oxygen (50). This activity may function in anaerobic respiration, but such a role has yet to be established (61). In addition, S. typhimurium (but not E. coli) has an anaerobically expressed membrane-bound thiosulfate reductase that is induced by thiosulfate and may play a role in anaerobic respiration (22). The gene or genes encoding this activity have not been characterized, but the phs locus is required for its expression and has been cloned (46).

S. typhimurium cysB mutants are also Cys⁺ when grown anaerobically, implying the existence of another regulatory gene that is expressed, or perhaps is active, only under anaerobic conditions (6). In K. aerogenes, a locus designated ORF-2 encodes a 316-residue protein with approximately 40% identity to CysB (166). If present in S. typhimurium, this gene could be responsible for anaerobic regulation of the cysteine operon.

S. typhimurium cysM mutants are Cys⁺ on sulfate under aerobic conditions but Cys^– when grown anaerobically (43), suggesting that anaerobic sulfate reduction may proceed through thiosulfate, which is then incorporated into S-sulfocysteine through the activity of the cysM product, O-acetylserine (thiol)-lyase-B.

CYSTEINYL-tRNA SYNTHETASE

The gene for E. coli cysteinyl-tRNA synthetase, cysS (13), encodes a 52-kDa polypeptide with the HIGH and KMSKS peptide motifs that identify it as a class I enzyme (2, 42, 55). Cysteinyl-tRNA synthetase is the smallest monomeric aminoacyl-tRNA synthetase of E. coli, and its sequence is closely related to those of the enzymes for methionine, leucine, isoleucine, and valine. This relatively small size and regions of homology with seryl-tRNA synthetase, a class II enzyme, have prompted speculation that cysteinyl-tRNA synthetase is closely related to a primordial aminoacyl-tRNA synthetase (2, 42). The enzyme forms small amounts of cysteine thiolactone from cysteinyl-tRNA^Cys via a nucleophilic attack on the cysteine carboxyl group by the sulfhydryl group (79). This activity may be the remnant of an ancient editing function that is no longer required because of the evolution of a high level of specificity for recognizing cysteine.

A single tRNA^Cys species has been identified in E. coli (117); the gene for this species is situated between those for a tRNA^Leu and a tRNA^Gly at 42 min on the chromosome (47, 92). This gene has been designated cysT but is distinct from the cysT for sulfate-thiosulfate permease. The discriminator base, U73, of tRNA^Cys is a major determinant for recognition by cysteinyl-tRNA synthetase, as are elements within the tertiary domain defined by the D stem-loop, the TΨC stem-loop, and the variable loop (91). Mutations in the anticodon wobble base also impair recognition, although changing the GCA cysteine anticodon to the GAA phenylalanine anticodon does not affect aminoacylation with cysteine.