Cysteine biosynthesis in Trichomonas vaginalis involves cysteine synthase utilizing O-phosphoserine

Trichomonas vaginalis is an early divergent eukaryote with many unusual biochemical features. It is an anaerobic protozoan parasite of humans that is thought to rely heavily on cysteine as a major redox buffer, because it lacks glutathione. We report here that for synthesis of cysteine from sulfide, T. vaginalis relies upon cysteine synthase. The enzyme (TvCS1) can use either O-acetylserine or O-phosphoserine as substrates. The K(m) values of the enzyme for sulfide are very low (0.02 mm), suggesting that the enzyme may be a means of ensuring that sulfide in the parasite is maintained at a low level. T. vaginalis appears to lack serine acetyltransferase, the source of O-acetylserine in many cells, but has a functional 3-phosphoglycerate dehydrogenase and an O-phosphoserine aminotransferase that together result in the production of O-phosphoserine, suggesting that this is the physiological substrate. TvCS1 can also use thiosulfate as substrate. Overall, TvCS1 has substrate specificities similar to those reported for cysteine synthases of Aeropyrum pernix and Escherichia coli, and this is reflected by sequence similarities around the active site. We suggest that these enzymes are classified together as type B cysteine synthases, and we hypothesize that the use of O-phosphoserine is a common characteristic of these cysteine synthases. The level of cysteine synthase in T. vaginalis is regulated according to need, such that parasites growing in an environment rich in cysteine have low activity, whereas exposure to propargylglycine results in elevated cysteine synthase activity. Humans lack cysteine synthase; therefore, this parasite enzyme could be an exploitable drug target.     

Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana

Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative effector O-acetylserine. We have determined an equilibrium dissociation constant of the cysteine synthase complex (K(D) = 25 +/- 4 x 10(-9) m), based on a reliable A + B <--> AB model of interaction. Analysis of dissociation kinetics in the presence of O-acetylserine revealed a half-maximal dissociation rate at 77 +/- 4 microm O-acetylserine and strong positive cooperativity for complex dissociation. The equilibrium of interaction was determined using an enzyme activity-based approach and yielded a K(m) value of 58 +/- 7 microm O-acetylserine. Both effector concentrations are in the range of intracellular O-acetylserine fluctuations and support a functional model that integrates effector-driven cysteine synthase complex dissociation as a regulatory switch for the biosynthetic pathway. The results show that BIAcore technology can be applied to obtain quantitative kinetic data of a hetero-oligomeric protein complex with enzymatic and regulatory function.     

Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors

The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.     

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.     

Three Arabidopsis genes encoding proteins with differential activities for cysteine synthase and beta-cyanoalanine synthase

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.     

Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharomyces pombe

Cysteine synthase catalyzes the formation of cysteine from O-acetylserine, and is the key enzyme for de novo cysteine biosynthesis in Schizosaccharomyces pombe. An examination of the S. pombe database revealed that two gene products are predicted to encode proteins homologous to eukaryotic cysteine synthases. Disruption of one of these candidates, cys1a+ (SPBC36.04), caused an obvious cysteine auxotrophy, while disruption of cys1b+ (SPAC3A12.17c) had no effect on the growth phenotype. Furthermore, overexpression of cys1b+ did not complement the cysteine auxotrophic phenotype of cys1a mutant cells. These results indicated that cys1a+, not cys1b+, primarily functions in the biosynthesis of cysteine in S. pombe cells. We constructed a bacterial-S. pombe shuttle vector containing cys1a+ as a selective marker gene. The combination of the cysteine auxotroph and new vector could be useful for the expression of a heterologous protein.     

Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) fromArabidopsis thaliana

Summary Cysteme synthase, the key enzyme for fixation of inorganic sulfide, catalyses the formation of cysteine from O-acetylserine and inorganic sulfide. Here we report the cloning of cDNAs encoding cysteine synthase isoforms fromArabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial and a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS-B; CSase B), respectively.AtCS-C andAtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (45.8 kD) and of 392 amino acids ( 41.8 kD), respectively. The deduced amino acid sequences of the mitochondrial and plastidic isoforms exhibit high homology even with respect to the presequences. The predicted presequence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed thatAtCS-C is higher expressed in roots than in leaves whereas the expression ofAtCS-B is stronger in leaves. Furthermore, gene expression of both genes was enhanced by sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of theAtCS-B gene is regulated by light. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) isoforms of cysteine synthase ofArabidopsis are able to complement a cysteine synthasedeficient mutant ofEscherichia coli unable to grow on minimal medium without cysteine, indicating synthesis of functional plant proteins in the bacterium. Two lines of evidence proved thatAtCS-C encodes a mitochondrial form of cysteine synthase; first, import ofin vitro translation products derived from AtCS-C in isolated intact mitochondria and second, Western blot analysis of mitochondria isolated from transgenic tobacco plants expressing AtCS-C cDNA/c-myc DNA fusion protein.Abbreviations CSase cysteine synthase The nucleotide sequence data reported will appear in the EMBL Database under the accession numbers X81973 forAtCS-C and X81698 forAtCS-B.     

The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction.

Serine acetyltransferase (SAT) catalyzes the rate-limiting step of cysteine biosynthesis in bacteria and plants and functions in association with O-acetylserine (thiol) lyase (OAS-TL) in the cysteine synthase complex. Very little is known about the structure and catalysis of SATs except that they share a characteristic C-terminal hexapeptide-repeat domain with a number of enzymatically unrelated acyltransferases. Computational modeling of this domain was performed for the mitochondrial SAT isoform from Arabidopsis thaliana, based on crystal structures of bacterial acyltransferases. The results indicate a left-handed parallel beta-helix consisting of beta-sheets alternating with turns, resulting in a prism-like structure. This model was challenged by site-directed mutagenesis and tested for a suspected dual function of this domain in catalysis and hetero-oligomerization. The bifunctionality of the SAT C-terminus in transferase activity and interaction with OAS-TL is demonstrated and discussed with respect to the putative role of the cysteine synthase complex in regulation of cysteine biosynthesis.     

Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma-synthesis with O-acetyl-L-homoserine and L-cysteine as substrates but not beta-synthesis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cystathionine beta-lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium. L-Cysteine and glutathione, both at 0.5 mM, added to the medium as the sole sulfur source repressed the synthesis of O-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions as a cysteine synthase. Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold. A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine gamma-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine gamma-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibited O-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T. Kosuge, D. Gao, and T. Hoshino, J. Biosci. Bioeng. 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine in T. thermophilus HB8.     

O-acetylserine sulfhydrylase and S-sulfocysteine synthase activities of Chromatium vinosum

Two proteins containing O-acetylserine sulfhydrylase activity were purified from Chromatium vinosum. Their separation was carried out by DE52 or Ecteola cellulose chromatography. While protein I with a molecular weight of 56,000 had only O-acetylserine sulfhydrylase activity, protein II with a molecular weight of 50,000 possessed S-sulfocysteine synthase activity in addition. It was not possible to separate the two activities of protein II by electrophoretic methods. The reaction rate of protein II with sulfide and O-acetylserine was twice as high as that with thiosulfate and O-acetylserine. When extracts of sulfate-grown cells were purified the major O-acetylserine activity was always associated with protein II. Regulatory and kinetic phenomena of the two activities were studied.     

Cysteine and S-sulfocysteine biosynthesis in phototrophic bacteria

Forteen species (17 strains) of phototrophic bacteria as well as one strain of Thiobacillus denitrificans were tested for cysteine synthase and S-sulfocysteine synthase. All strains contain cysteine synthase active with O-acetylserine; only the Chromatiaceae, two species of the Rhodospirillaceae and T. denitrificans contain S-sulfocysteine synthase. In six species repression by different sulfur compounds in the medium was studied. In Chromatium vinosum, cysteine synthase was found to be constitutive, while in the Rhodospirillaceae tested the enzyme is repressed by sulfide. Thiosulfate had a derepressive effect in Rhodopseudomonas globiformis but strongly repressed cysteine synthase in R. sulfidophila and R. palustris. Cysteine had only moderate effects with the species tested.     

Glutamate synthase. Properties of the glutamine-dependent activity.

Properties of glutamine-dependent glutamate synthase have been investigated using homogeneous enzyme from Escherichia coli K-12. In contrast to results with enzyme from E. coli strain B (Miller, R. E., and Stadtman, E. R. (1972) J. Biol. Chem. 247, 7407-7419), this enzyme catalyzes NH3-dependent glutamate synthase activity. Selective inactivation of glutamine-dependent activity was obtained by treatment with the glutamine analog. L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone). Inactivation by chloroketone exhibited saturation kinetics; glutamine reduced the rate of inactivation and exhibited competitive kinetics. Iodoacetamide, other alpha-halocarbonyl compounds, and sulfhydryl reagents gave similar selective inactivation of glutamine-dependent activity. Saturation kinetics were not obtained for inactivation by iodoacetamide but protection by glutamine exhibited competitive kinetics. The stoichiometry for alkylation by chloroketone and iodoacetamide was approximately 1 residue per protomer of molecular weight approximately 188,000. The single residue alkylated with iodo [1-14C]acetamide was identified as cysteine by isolation of S-carboxymethylcysteine. This active site cysteine is in the large subunit of molecular weight approximately 153,000. The active site cysteine was sensitive to oxidation by H2O2 generated by autooxidation of reduced flavin and resulted in selective inactivation of glutamine-dependent enzyme activity. Similar to other glutamine amidotransferases, glutamate synthase exhibits glutaminase activity. Glutaminase activity is dependent upon the functional integrity of the active site cysteine but is not wholly dependent upon the flavin and non-heme iron. Collectively, these results demonstrate that glutamate synthase is similar to other glutamine amidotransferases with respect to distinct sites for glutamine and NH3 utilization and in the obligatory function of an active site cysteine residue for glutamine utilization.     

Cysteine synthase (CysM) of Mycobacterium tuberculosis is an O-phosphoserine sulfhydrylase: evidence for an alternative cysteine biosynthesis pathway in mycobacteria

The biosynthesis of cysteine is a crucial metabolic pathway supplying a building block for de novo protein synthesis but also a reduced thiol as a component of the oxidative defense mechanisms that appear particularly vital in the dormant state of Mycobacterium tuberculosis. We here show that the cysteine synthase CysM is, in contrast to previous annotations, an O-phosphoserine-specific cysteine synthase. CysM belongs to the fold type II pyridoxal 5'-phosphate-dependent enzymes, as revealed by the crystal structure determined at 2.1-angstroms resolution. A model of O-phosphoserine bound to the enzyme suggests a hydrogen bonding interaction of the side chain of Arg220 with the phosphate group as a key feature in substrate selectivity. Replacement of this residue results in a significant loss of specificity for O-phosphoserine. Notably, reactions with sulfur donors are not affected by the amino acid replacement. The specificity of CysM toward O-phosphoserine together with the previously established novel mode of sulfur delivery via thiocarboxylated CysO (Burns, K. E., Baumgart, S., Dorrestein, P. C., Zhai, H., McLafferty, F. W., and Begley, T. P. (2005) J. Am. Chem. Soc. 127, 11602-11603) provide strong evidence for an O-phosphoserine-based cysteine biosynthesis pathway in M. tuberculosis that is independent of both O-acetylserine and the sulfate reduction pathway. The existence of an alternative biosynthetic pathway to cysteine in this pathogen has implications for the design strategy aimed at inhibition of this metabolic route.     

Role of a cysteine synthase in Staphylococcus aureus

The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5alpha. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid- or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.     

S-sulfocysteine synthase function in sensing chloroplast redox status

The minor chloroplastic O-acetylserine(thiol)lyase isoform encoded by the CS26 gene in Arabidopsis thaliana has been described as an S-sulfocysteine synthase enzyme that plays an important role in chloroplast function. This enzyme is located in the thylakoid lumen, and its S-sulfocysteine activity is essential for the proper photosynthetic performance of the chloroplast under long-day growth conditions. Based on the present knowledge of this enzyme, we suggest that S-sulfocysteine synthase functions as a protein sensor to detect the accumulation of thiosulfate as a result of the inadequate detoxification of reactive oxygen species generated under conditions of excess light to produce the S-sulfocysteine molecule that triggers protection mechanisms of the photosynthetic apparatus.     

The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase

The biosynthesis of cysteine in bacteria and plants is carried out by a two-step pathway, catalyzed by serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS; O-acetylserine [thiol] lyase). The aerobic form of OASS forms a tight bienzyme complex with SAT in vivo, termed cysteine synthase. We have determined the crystal structure of OASS in complex with a C-terminal peptide of SAT required for bienzyme complex formation. The binding site of the peptide is at the active site of OASS, and its C-terminal carboxyl group occupies the same anion binding pocket as the alpha-carboxylate of the O-acetylserine substrate of OASS. These results explain the partial inhibition of OASS by SAT on complex formation as well as the competitive dissociation of the complex by O-acetylserine.     

Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase.

1. Serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase were purified from Paracoccus denitrificans strain 8944. 2. Serin transacetylase was purified 150-fold. The enzyme has a pH optimum between 7.5 and 8.0, is specific for L-serine and is inhibited by sulphydryl-group reagents. The apparent Km values for serine and acetyl-CoA are 4.0 - 10(-4) and 1.0 - 10(-4) M, respectively. Serine transacetylase is strongly inhibited by cysteine. 3. O-Acetylserine sulphydrylase was purified 450-fold. The enzymes has a sharp pH optimum at pH 7.5. In addition to catalysing the synthesis of cysteine, O-acetylserine sulphydrylase catalyses the synthesis of selenocysteine from O-acetylserine and selenide. The Km values for sulphide and O-acetylserine are 2.7 - 10(-3) and 1.25 - 10(-3) M, respectively. The enzyme was stimulated by pyridoxal phosphate and was inhibited by cystathionine, homocysteine and methionine. 4. beta-Cystathionase was purified approx. 50-fold. beta-Cystathionase has a pH optimum between pH 9.0 and 9.5, is sensitive to sulphydryl-group reagents, required pyridoxal phosphate for maximum activity and has an apparent Km for cystathionine of 4.2 - 10 (-3) M. beta-Cystathionase also catalyses the release of keto acid from lanthionine, djenkolic acid and cystine. Cysteine, O-acetylserine, homocysteine and glutathione strongly inhibit beta-cystathionase activity and homocysteine and methionine represses enzyme activity. 5. O-Acetylserine lyase was identified in crude extracts of Paracoccus denitrificans. The enzyme is specific for O-acetyl-L-serine, requires pyridoxal phosphate and is inhibied by KCN and hydroxylamine. The enzyme has a high Km value for O-acetylserine (50--100 mM).     

YfiK from Escherichia coli promotes export of O-acetylserine and cysteine

yfiK was discovered as a gene augmenting cysteine production when it was overexpressed in an industrial Escherichia coli production strain. The gene product is an integral membrane protein with about six predicted transmembrane helices; it belongs to the RhtB family of export proteins. YfiK overproduction from a plasmid leads to drastic and parallel secretion of O-acetylserine and cysteine into the medium but only when the organism possesses a serine transacetylase that is feedback insensitive to cysteine. Externally provided O-acetylserine obviated this requirement for cysteine secretion both in the yfiK-carrying transformant and in the wild type. A DeltayfiK mutant did not show any phenotype, and it exported O-acetylserine and cysteine when transformed with a plasmid carrying ydeD, a previously characterized, alternate O-acetylserine/cysteine exporter. Since a ydeD-yfiK double mutant showed the same pattern, it appears that YfiK and YdeD act independently. The necessity for the cell to regulate the size of the internal pool of O-acetylserine via synthesis of exporter proteins could be connected to the fact that this compound (when supplied externally) inhibits growth. Overexpression of either ydeD or yfiK leads to alleviation of this inhibition paralled by increased resistance to azaserine, which is an analog of O-acetylserine.