Inhibition of O-acetylserine (thiol) lyase as a promising new mechanism of action for herbicides.

Enzymes of the sulfur assimilation pathway of plants have been identified as potential targets for herbicide development, given their crucial role in synthesizing amino acids, coenzymes, and various sulfated compounds. In this pathway, O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) catalyzes the synthesis of L-cysteine through the incorporation of sulfate into O-acetylserine (OAS). This study used an in silico approach to select seven inhibitors for OAS-TL. The in silico experiments revealed that S-benzyl-L-cysteine (SBC) had a better docking score (-7.0 kcal mol-1) than the substrate OAS (-6.6 kcal mol-1), indicating its suitable interaction with the active site of the enzyme. In vitro experiments showed that SBC is a non-competitive inhibitor of OAS-TL from Arabidopsis thaliana expressed heterologously in Escherichia coli, with a Kic of 4.29 mM and a Kiu of 5.12 mM. When added to the nutrient solution, SBC inhibited the growth of maize and morning glory weed plants due to the reduction of L-cysteine synthesis. Remarkably, morning glory was more sensitive than maize. As proof of its mechanism of action, L-cysteine supplementation to the nutrient solution mitigated the inhibitory effect of SBC on the growth of morning glory. Taken together, our data suggest that reduced L-cysteine synthesis is the primary cause of growth inhibition in maize and morning glory plants exposed to SBC. Furthermore, our findings indicate that inhibiting OAS-TL could potentially be a novel approach for herbicidal action.

A molecular switch in sulfur metabolism to reduce arsenic and enrich selenium in rice grain.

Rice grains typically contain high levels of toxic arsenic but low levels of the essential micronutrient selenium. Anthropogenic arsenic contamination of paddy soils exacerbates arsenic toxicity in rice crops resulting in substantial yield losses. Here, we report the identification of the gain-of-function arsenite tolerant 1 (astol1) mutant of rice that benefits from enhanced sulfur and selenium assimilation, arsenic tolerance, and decreased arsenic accumulation in grains. The astol1 mutation promotes the physical interaction of the chloroplast-localized O-acetylserine (thiol) lyase protein with its interaction partner serine-acetyltransferase in the cysteine synthase complex. Activation of the serine-acetyltransferase in this complex promotes the uptake of sulfate and selenium and enhances the production of cysteine, glutathione, and phytochelatins, resulting in increased tolerance and decreased translocation of arsenic to grains. Our findings uncover the pivotal sensing-function of the cysteine synthase complex in plastids for optimizing stress resilience and grain quality by regulating a fundamental macronutrient assimilation pathway.

Crystal structure of O-Acetylserine sulfhydralase (OASS) isoform 3 from Entamoeba histolytica: Pharmacophore-based virtual screening and validation of novel inhibitors.

The l-cysteine is crucial for growth, survival, defense against oxidative stress, and pathogenesis of Entamoeba histolytica. The de novo biosynthesis of l-cysteine in E. histolytica, has a two-step pathway, where O-acetylserine sulfhydrylase (OASS) catalyses the last step by converting OAS to l-cysteine. This pathway is absent in humans and hence represents a promising target for novel therapeutics. E. histolytica expresses three isoforms of OASS and knockdown studies showed the importance of these enzymes for the survival of the pathogen. Here, we report the crystal structure of OASS isoform 3 from E. histolytica to 1.54 Å resolution. The active site geometries and kinetics of EhOASS3 and EhOASS1 structures were found to be very similar. Small-molecule libraries were screened against EhOASS3 and compounds were shortlisted based on the docking scores. F3226-1387 showed best inhibition with IC50 of 38 µM against EhOASS3 and was able to inhibit the growth of the organism to 72%.

Higher Novel L-Cys Degradation Activity Results in Lower Organic-S and Biomass in Sarcocornia than the Related Saltwort, Salicornia.

Salicornia and Sarcocornia are almost identical halophytes whose edible succulent shoots hold promise for commercial production in saline water. Enhanced sulfur nutrition may be beneficial to crops naturally grown on high sulfate. However, little is known about sulfate nutrition in halophytes. Here we show that Salicornia europaea (ecotype RN) exhibits a significant increase in biomass and organic-S accumulation in response to supplemental sulfate, whereas Sarcocornia fruticosa (ecotype VM) does not, instead exhibiting increased sulfate accumulation. We investigated the role of two pathways on organic-S and biomass accumulation in Salicornia and Sarcoconia: the sulfate reductive pathway that generates Cys and l-Cys desulfhydrase that degrades Cys to H2S, NH3, and pyruvate. The major function of O-acetyl-Ser-(thiol) lyase (OAS-TL; EC 2.5.1.47) is the formation of l-Cys, but our study shows that the OAS-TL A and OAS-TL B of both halophytes are enzymes that also degrade l-Cys to H2S. This activity was significantly higher in Sarcocornia than in Salicornia, especially upon sulfate supplementation. The activity of the sulfate reductive pathway key enzyme, adenosine 5'-phosphosulfate reductase (APR, EC 1.8.99.2), was significantly higher in Salicornia than in Sarcocornia These results suggest that the low organic-S level in Sarcocornia is the result of high l-Cys degradation rate by OAS-TLs, whereas the greater organic-S and biomass accumulation in Salicornia is the result of higher APR activity and low l-Cys degradation rate, resulting in higher net Cys biosynthesis. These results present an initial road map for halophyte growers to attain better growth rates and nutritional value of Salicornia and Sarcocornia.

CysK Plays a Role in Biofilm Formation and Colonization by Vibrio fischeri.

A biofilm, or a matrix-embedded community of cells, promotes the ability of the bacterium Vibrio fischeri to colonize its symbiotic host, the Hawaiian squid Euprymna scolopes. Biofilm formation and colonization depend on syp, an 18-gene polysaccharide locus. To identify other genes necessary for biofilm formation, we screened for mutants that failed to form wrinkled colonies, a type of biofilm. We obtained several with defects in genes required for cysteine metabolism, including cysH, cysJ, cysK, and cysN. The cysK mutant exhibited the most severe wrinkling defect. It could be complemented with a wild-type copy of the cysK gene, which encodes O-acetylserine sulfhydrolase, or by supplementing the medium with additional cysteine. None of a number of other mutants defective for biosynthetic genes negatively impacted wrinkled colony formation, suggesting a specific role for CysK. CysK did not appear to control activation of Syp regulators or transcription of the syp locus, but it did influence production of the Syp polysaccharide. Under biofilm-inducing conditions, the cysK mutant retained the same ability as that of the parent strain to adhere to the agar surface. The cysK mutant also exhibited a defect in pellicle production that could be complemented by the cysK gene but not by cysteine, suggesting that, under these conditions, CysK is important for more than the production of cysteine. Finally, our data reveal a role for cysK in symbiotic colonization by V. fischeri. Although many questions remain, this work provides insights into additional factors required for biofilm formation and colonization by V. fischeri.

Molecular dynamic simulation and inhibitor prediction of cysteine synthase structured model as a potential drug target for trichomoniasis.

In our presented research, we made an attempt to predict the 3D model for cysteine synthase (A2GMG5_TRIVA) using homology-modeling approaches. To investigate deeper into the predicted structure, we further performed a molecular dynamics simulation for 10 ns and calculated several supporting analysis for structural properties such as RMSF, radius of gyration, and the total energy calculation to support the predicted structured model of cysteine synthase. The present findings led us to conclude that the proposed model is stereochemically stable. The overall PROCHECK G factor for the homology-modeled structure was -0.04. On the basis of the virtual screening for cysteine synthase against the NCI subset II molecule, we present the molecule 1-N, 4-N-bis [3-(1H-benzimidazol-2-yl) phenyl] benzene-1,4-dicarboxamide (ZINC01690699) having the minimum energy score (-13.0 Kcal/Mol) and a log P value of 6 as a potential inhibitory molecule used to inhibit the growth of T. vaginalis infection.

Mutation of L-2,3-diaminopropionic acid synthase genes blocks staphyloferrin B synthesis in Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus synthesizes two siderophores, staphyloferrin A and staphyloferrin B, that promote iron-restricted growth. Previous work on the biosynthesis of staphyloferrin B has focused on the role of the synthetase enzymes, encoded from within the sbnA-I operon, which build the siderophore from the precursor molecules citrate, alpha-ketoglutarate and L-2,3-diaminopropionic acid. However, no information yet exists on several other enzymes, expressed from the biosynthetic cluster, that are thought to be involved in the synthesis of the precursors (or synthetase substrates) themselves. RESULTS: Using mutants carrying insertions in sbnA and sbnB, we show that these two genes are essential for the synthesis of staphyloferrin B, and that supplementation of the growth medium with L-2,3-diaminopropionic acid can bypass the block in staphyloferrin B synthesis displayed by the mutants. Several mechanisms are proposed for how the enzymes SbnA, with similarity to cysteine synthase enzymes, and SbnB, with similarity to amino acid dehydrogenases and ornithine cyclodeaminases, function together in the synthesis of this unusual nonproteinogenic amino acid L-2,3-diaminopropionic acid. CONCLUSIONS: Mutation of either sbnA or sbnB result in abrogation of synthesis of staphyloferrin B, a siderophore that contributes to iron-restricted growth of S. aureus. The loss of staphyloferrin B synthesis is due to an inability to synthesize the unusual amino acid L-2,3-diaminopropionic acid which is an important, iron-liganding component of the siderophore structure. It is proposed that SbnA and SbnB function together as an L-Dap synthase in the S. aureus cell.

Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration.

A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.

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.

Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma.

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.

Purification and characterization of beta-cyanoalanine synthase and cysteine synthases from potato tubers: are beta-cyanoalanine synthase and mitochondrial cysteine synthase same enzyme?

beta-Cyanoalanine synthase (CAS; EC 4.4.1.9) and two kinds of cysteine synthases (CS; EC 4.2.99.8) have been purified from the particulate fraction of potato tubers. By DEAE Sephacel and Resource PHE chromatography, CAS activity was separated from two CS activities, designated as CS-1 and CS-2. The molecular masses of CAS, CS-1 and CS-2 were estimated to be 37, 39 and 34 kDa, respectively, by SDS-PAGE analysis. The purified CAS had CS activity, and both CS-1 and CS-2 had CAS activity. However, CAS and CSs had significant differences in kinetic characters. The antibody raised against purified CAS discriminated CAS from CSs, whereas the antibody raised against purified CS-2 recognized CS-1 and CS-2 but not CAS. The molecular mass and the partial amino acid sequence of CS-2 were similar to those of the cytosolic CS of potato, whereas the molecular mass of CS-1 was similar to that of the plastidic CS. The partial amino acid sequence of CAS was similar to those of CS isozymes, especially the mitochondrial CS isolated from spinach.

Cloning and characterization of the gene encoding O-acetylserine lyase from Streptococcus suis.

We have cloned and sequenced a gene encoding O-acetylserine lyase from Streptococcus suis. The gene encodes a protein of 309 amino acids with a calculated molecular mass of 32,038 Da. The deduced amino acid sequence showed more extensive similarities to the CysK proteins than to the CysM proteins of other bacteria. The cloned gene was inserted into a pTrcHisB histidine hexamer expression vector. A 38-kDa fusion protein was expressed in a cysMK auxotrophic mutant of Salmonella typhimurium and complemented the auxotrophic properties of the mutant. Furthermore, the transformants could grow in minimal defined media supplemented with not only sulfide but also thiosulfate as a sole sulfur source. These data indicated that the cloned gene encodes a protein that was a functional homolog of the CysM in S. typhimurium.

Cysteine biosynthesis in Chlamydomonas reinhardtii. Molecular cloning and regulation of O-acetylserine(thiol)lyase.

A cDNA, Cys1ACr, encoding an isoform of O-acetylserine(thiol) lyase has been isolated from Chlamydomonas reinhardtii, using a PCR-based approach. The inclusion of dimethylsulfoxide in the PCR reaction has been demonstrated to be essential for the correct amplification of C. reinhardtii templates with complex secondary structures caused by a high G + C content. The deduced amino acid sequence exhibited highest similarity with plant O-acetylserine(thiol)lyase isoforms, indicating that the C. reinhardtii enzyme was structurally more similar to higher plant O-acetylserine(thiol)lyase than to the corresponding prokaryotic enzymes. The N-terminal extension present in Cys1ACr showed several characteristics of an organellar transit peptide, with a length typical for C. reinhardtii. Southern blot analysis suggested that the C. reinhardtii genome may contain a single copy of the organellar O-acetylserine(thiol)lyase gene. O-acetylserine(thiol)lyase activity was strongly induced by sulfur-deficient conditions (up to sevenfold the level observed in a sulfur-repleted cell culture) and required the presence of a nitrogen source. Northern blot analysis showed a different pattern of regulation of Cys1ACr to that observed at the activity level. To obtain an increase of transcript abundance a longer period of sulfur limitation was required, reaching a maximum level of approximately threefold Cys1ACr mRNA when compared with the level of a sulfate-grown culture.

The path of unspecific incorporation of selenium in Escherichia coli.

The path of unspecific selenium incorporation into proteins was studied in Escherichia coli mutants blocked in the biosynthesis of cysteine and methionine or altered in its regulation. Selenium incorporation required all enzymatic steps of cysteine biosynthesis except sulfite reduction, indicating that intracellular reduction of selenite occurs nonenzymatically. Cysteine (but not methionine) supplementation prevented unspecific incorporation of selenium by repressing cysteine biosynthesis. On the other hand, when the biosynthesis of cysteine was derepressed in regulatory mutants, selenium was incorporated to high levels. These findings and the fact that methionine auxotrophic strains still displayed unspecific incorporation show that selenium incorporation into proteins in E. coli occurs mainly as selenocysteine. These findings also provide information on the labeling conditions for incorporating 75Se only and specifically into selenoproteins.

Enzymatic synthesis of two isoxazolylalanine isomers by cysteine synthases in Lathyrus species.

Two isoxazolylalanine isomers, beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA, 1) and beta-(isoxazolin-5-on-4-yl)-L- alanine (TAN-950A, 2) were confirmed to be derived from O-acetyl-L-serine (OAS) and isoxazolin-5-one by cysteine synthases (CSases) with a different ratio in different plant parts. Some properties of this enzyme in the biosynthesis of both isomers are described.

Purification and characterization of O-acetylserine sulfhydrylase isoenzymes from Datura innoxia.

Three isoenzyme forms (designated A, B, and C) of O-acetylserine sulfhydrylase were purified from Datura innoxia suspension cultures. Isoenzyme A is the most abundant form, comprising 45-60% of the total activity. Isoenzymes C and B comprise 35-40% and 10-20% of the activity, respectively. The specific activities of the purified isoenzymes are similar (870-893 mumol of cysteine/min/mg of protein). Molecular masses for isoenzymes A, B, and C, estimated by analytical size exclusion high performance liquid chromatography, are 63, 86, and 63 kDa, respectively. Isoenzymes A and B are homodimers; isoenzyme C is a heterodimer. Spectral analysis indicates that these isoenzymes possess a pyridoxal 5'-phosphate cofactor that binds the O-acetylserine substrate. Binding is reversible by addition of the sulfide substrate. The O-acetylserine sulfhydrylase isoenzymes are active over a broad temperature range, with maximum activity between 42 and 58 degrees C. They are active only between pH 7 and 8, with optimal activity at pH 7.6. Kinetic analysis indicates these enzymes are allosterically regulated and exhibit positive cooperativity with respect to both substrates. They are inhibited by sulfide concentrations above 200 microM. The kinetic analysis together with the physical and spectrophotometric characteristics indicate that the O-acetylserine sulfhydrylase enzymes have two active sites.

Cysteine synthase from Capsicum annuum chromoplasts. Characterization and cDNA cloning of an up-regulated enzyme during fruit development.

Cysteine synthase (O-acetylserine sulfhydrylase) has been purified to homogeneity from bell pepper (Capsicum annuum) fruit chromoplasts. This enzyme consists of two subunits of 35 kDa. Immunocytochemical localization experiments confirmed the plastid location of this enzyme. A full-length cDNA was isolated from an expression library of C. annuum. The deduced peptide sequence revealed high similarity between the C. annuum cysteine synthase and its bacterial counterparts. In vitro transcription and translation of the cDNA and subsequent import experiments demonstrated that the encoded cysteine synthase is located in the plastids. The steady-state level of the cysteine synthase mRNA is almost constant in dark-grown hypocotyls, leaves, and fruits. However, a slight increase in this mRNA level was detected during fruit development (when the 25 S rRNA was taken as an internal standard). Similarly, the cysteine synthase activity in plastids was found to increase during fruit development and reaches the highest levels in the chromoplasts of red fruits. To address the physiological role of this phenomenon, we have shown that cysteine is engaged in the active metabolism of glutathione. Thus, in connection with the previous demonstration of an active tocopherol metabolism, it is concluded that differentiation of chloroplast to chromoplast in C. annuum involves an active synthesis of potential antioxidants or redox modulators.

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).