O-alkylhomoserine synthesis catalyzed by O-acetylhomoserine sulfhydrylase in microorganisms.

An enzyme that can synthesize O-alkylhomoserine from alcohols and O-acetylhomoserine was purified from Corynebacterium acetophilum. The enzyme was found to be identical to O-acetylhomoserine sulfhydrylase; a preparation that appeared homogeneous on polyacrylamide gel electrophoresis showed both O-alkylhomoserine-synthesizing and O-acetylhomoserine sulfhydrylase activities. Its molecular weight was determined to be about 220,000, and it consisted of two subunits. Its pH and temperature optima for the two reactions were the same. Besides catalyzing the formation of homocysteine from O-acetylhomoserine and sulfide, it also catalyzed the syntheses of O-alkylhomoserines corresponding to the alcohols added form O-acetylhomoserine and ethyl alcohol, n-propylalcohol, n-butyl alcohol, methyl alcohol, and n-pentyl alcohol, its activities with these alcohols decreasing in that order. L-Homoserine, O-succinylhomoserine, and O-acetylserine reacted with sulfide. O-ethylhomoserine, O-acetylthreonine, O-succinylhomoserine, and O-acetylserine inhibited both enzyme activities. O-acetylhomoserine sulfhydrylase purified from Saccharomyces cerevisiae also showed O-alkylhomoserine-synthesizing activity. Thus, O-acetylhomoserine sulfhydrylase seems to catalyze O-alkylhomoserine synthesis in the presence of appropriate concentrations of alcohol and O-acetylhomoserine in microorganisms.     

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.     

Cystathionine Synthesis in Yeast: an Alternative Pathway for Homocysteine Biosynthesis

Cystathionine synthesis from O-acetylhomoserine and cysteine has been demonstrated in yeast extracts for the first time. The activity is less than that of O-acetylhomoserine sulfhydrylase, but it is higher than that reported for homoserine O-transacetylase and therefore should not be growth limiting. Cystathionine synthase seems to share the regulatory properties of the sulfhydrylase, and both activities are missing from the methionine auxotroph Saccharomyces cerevisiae EY9, suggesting that both reactions are catalyzed by the same enzyme. However, cystathionine synthase activity was lost during purification of the sulfhydrylase, suggesting that the two reactions may be catalyzed by separate enzymes. Since previous studies have shown that yeast extracts can catalyze the cleavage of cystathionine to homocysteine, our results show the existence of two complete alternate pathways for homocysteine biosynthesis in yeast. Which of these is the major physiological pathway remains to be determined.     

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.     

Role of hydrosulfide ions (HS-) in methylmercury resistance in Saccharomyces cerevisiae.

Methylmercury-resistant mutants were obtained from Saccharomyces cerevisiae. They were divided into two complementation groups, met2 (homoserine O-acetyltransferase deficiency) and met15 (enzyme deficiency unknown), as reported previously. It was found that met15 was allelic to met17 (O-acetylserine and O-acetylhomoserine sulfhydrylase deficiency). Methylmercury toxicity was counteracted by exogenously added HS-, and both met2 and met17 (met15) mutants overproduced H2S. On the basis of these results, we conclude that met2 and met17 (met15) cause accumulation of hydrosulfide ions in the cell and that the increased level of hydrosulfide is responsible for detoxification of methylmercury.     

Methionine Biosynthesis in Lemna: STUDIES ON THE REGULATION OF CYSTATHIONINE gamma-SYNTHASE, O-PHOSPHOHOMOSERINE SULFHYDRYLASE, AND O-ACETYLSERINE SULFHYDRYLASE

Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine gamma-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine gamma-synthase, it was observed that (a) adding external methionine (2 mum) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 muml-aminoethoxyvinylglycine or with 36 mum lysine plus 4 mum threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine gamma-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase.The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine gamma-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine gamma-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of O-acetylserine sulfhydrylase provides an explanation in molecular terms for transsulfuration, and not direct sulfhydration, being the dominant pathway for homocysteine biosynthesis.     

Role of hydrosulfide ions (HS-) in methylmercury resistance in Saccharomyces cerevisiae.

Methylmercury-resistant mutants were obtained from Saccharomyces cerevisiae. They were divided into two complementation groups, met2 (homoserine O-acetyltransferase deficiency) and met15 (enzyme deficiency unknown), as reported previously. It was found that met15 was allelic to met17 (O-acetylserine and O-acetylhomoserine sulfhydrylase deficiency). Methylmercury toxicity was counteracted by exogenously added HS-, and both met2 and met17 (met15) mutants overproduced H2S. On the basis of these results, we conclude that met2 and met17 (met15) cause accumulation of hydrosulfide ions in the cell and that the increased level of hydrosulfide is responsible for detoxification of methylmercury.     

Regulation of O-acetylserine sulfhydrylase B by L-cysteine in Salmonella typhimurium.

A technique based on resistance to azaserine was used to isolate mutants lacking O-acetylserine sulfhydrylase B, one of two enzymes in Salmonella typhimurium capable of synthesizing L-cysteine from O-acetyl-L-serine and sulfide. The mutant locus responsible for this defect has been designated cysM, and genetic mapping suggests that cysM is very close to and perhaps contiguous with cysA. Strains lacking either O-acetylserine sulfhydrylase B or the second sulfhydrylase, O-acetylserine sulfhydrylase A (coded for by cysK), are cysteine prototrophs, but cysK cysM double mutants were found to require cysteine for growth. O-Acetylserine sulfhydrylase B was depressed by growth on a poor sulfur source, and depression was dependent upon both a functional cysB regulatory gene product and the internal inducer of the cysteine biosynthetic pathway, O-acetyl-L-serine. Furthermore, a cysBc strain, in which other cysteine biosynthetic enzymes cannot be fully repressed by growth on L-cystine, was found to be constitutive for O-acetylserine sulfhydrylase B as well. Thus O-acetylserine sulfhydrylase B is regulated by the same factors that control the expression of O-acetylserine sulfhydrylase A and other activities of the cysteine regulon. It is not clear why S. typhimurium has two enzymes whose physiological function appears to be to catalyze the same step of L-cysteine biosynthesis.     

Sulfite formation by wine yeasts

Four strains of wine yeasts of two different species (Saccharomyces cerevisiae var. ellipsoideus and S. bayanus) were investigated with respect to regulation of NADPH- and benzyl viologen-dependent sulfite reductases by various sulfur sources. The enzyme activity was followed over a growth period of 96 h. The low sulfite-producing strains showed an increased biosynthesis of NADPH-dependent sulfite reductase during the exponential growth phase in the presence of sulfate, sulfite and djencolic-acid. This increase was not observed in the high sulfite-producing strains. Methionine and cysteine prevented this derepression. At the end of the exponential growth phase, enzyme biosynthesis was repressed again, presumably by sulfur-containing amino acids which were produced during growth. The regulatory influence of the various sulfur sources on benzyl viologen dependent sulfitereductase activity is obviously much weaker.Abbreviation BV benzyl viologen     

Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Characterization of Cystathionine β-Synthase TtCbs1 and Cysteine Synthase TtCsa1 Involved in Cysteine Biosynthesis in Tetrahymena thermophila

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β-synthase and cystathionine γ-lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5′-phosphate-dependent enzymes, cystathionine β-synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N-terminal heme binding domain, catalytic domain, and C-terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His-TtCbs1 and His-TtCsa1 were expressed and purified in vitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high K_m for O-acetylserine and low K_m for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.     

Studies of l-Cysteine Biosynthetic Enzymes in Phaseolus vulgaris L

In higher plants the biosynthesis of l-cysteine from l-serine, acetylCoA, and sulfide requires serine transacetylase and O-acetylserine sulfhydrylase. The distribution of these enzymes in kidney bean (Phaseolus vulgaris L. cv. Red Kidney) seedlings was determined. Between one-third and two-thirds of the serine transacetylase activity was associated with mitochondria, whereas all of the O-acetyl-serine sulfhydrylase activity was present in the soluble fraction of cell homogenates. In a 14-day plant approximately two-thirds of the O-acetylserine sulfhydrylase activity and approximately one-half of the serine transacetylase activity was found in the leaves.     

Sulfate assimilation in Rhodopseudomonas globiformis

Rhodopseudomonas globiformis is able to grow on sulfate as sole source of sulfur, but only at concentrations below 1 mM. Good growth was observed with thiosulfate, cysteine or methionine as sulfur sources. Tetrathionate supported slow growth. Sulfide and sulfite were growth inhibitory. Growth inhibition by higher sulfate concentrations was overcome by the addition of O-acetylserine, which is known as derepressor of sulfate-assimilating enzymes, and by reduced glutathione. All enzymes of the sulfate assimilation pathway. ATP-sulfurylase, adenylylphosphate-sulfotransferase, thiosulfonate reductase and O-acetylserine sulfhydrylase are present in R. globiformis. Sulfate was taken up by the cells and the sulfur incorporated into the amino acids cysteine, methionine and homocysteine. It is concluded, that the failure of R. globiformis to grow on higher concentrations of sulfate is caused by disregulation of the sulfate assimilation pathway. Some preliminary evidence for this view is given in comparing the activities of some of the involved enzymes after growth on different sulfur sources and by examining the effect of O-acetylserine on these activities.Abbreviations DTE dl-dithioerythritol - APS adenosine 5-phosphosulfate, adenylyl sulfate - PAPS 3-phosphoadenosine 5-phosphosulfate, 3-phosphoadenylylsulfate     

Cloning of the E. coli O-acetylserine sulfhydrylase gene: ability of the clone to produce a mutagenic product from azide and O-acetylserine

The gene coding for O-acetylserine sulfhydrylase (OASS) from E. coli K12 was cloned into the vector pBR322 plasmid and expressed in a cysk mutant strain of E. coli that is deficient in O-acetylserine sulfhydrylase (OASS-). The clone containing the OASS gene was selected by using tetracycline-ammonium bismuth citrate medium. Retransformation of the hybrid plasmid into competent cysk mutant cells resulted in the recovery of a clone containing normal levels of O-acetylserine sulfhydrylase. Negative selection of retransformed cysk cells on 1,2,4-triazole plates resulted in the complete inhibition of growth indicating the presence of a functional OASS gene. The ability of the new clone to convert azide to its mutagenic metabolite was tested. Cultures of the clone cells containing significant levels of OASS activity were able to produce a mutagenic product from azide and O-acetylserine as tested on Salmonella typhimurium TA1530. This cloning method could be applied also to clone the same gene from eukaryotic sources.     

Purification and initial kinetic characterization of different forms of o-acetylserine sulfhydrylase from seedlings of two species of phaseolus

Purification of O-acetylserine sulfhydrylase (OASS) from seedlings of two species of Phaseolus reveals the presence in both species of two forms of this enzyme. The isolation and purification procedure gives purification of 7- to 160-fold for individual isoenzymes with specific activities ranging from 33 IU mg⁻¹ to 775 IU mg⁻¹ protein.     

Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy

Serine acetyltransferase is a key enzyme in the sulfur assimilation pathway of bacteria and plants, and is known to form a bienzyme complex with O-acetylserine sulfhydrylase, the last enzyme in the cysteine biosynthetic pathway. The biological function of the complex and the mechanism of reciprocal regulation of the constituent enzymes are still poorly understood. In this work the effect of complex formation on the O-acetylserine sulfhydrylase active site has been investigated exploiting the fluorescence properties of pyridoxal 5'-phosphate, which are sensitive to the cofactor microenvironment and to conformational changes within the protein matrix. The results indicate that both serine acetyltransferase and its C-terminal decapeptide bind to the alpha-carboxyl subsite of O-acetylserine sulfhydrylase, triggering a transition from an open to a closed conformation. This finding suggests that serine acetyltransferase can inhibit O-acetylserine sulfhydrylase catalytic activity with a double mechanism, the competition with O-acetylserine for binding to the enzyme active site and the stabilization of a closed conformation that is less accessible to the natural substrate.     

MET17 and hydrogen sulfide formation in Saccharomyces cerevisiae

Commercial isolates of Saccharomyces cerevisiae differ in the production of hydrogen sulfide (H(2)S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing the MET17 gene, which encodes the bifunctional O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H(2)S release. Overexpression of MET17 resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H(2)S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H(2)S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H(2)S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H(2)S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H(2)S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H(2)S production and release.     

Activation of sodium cyanide to a toxic but non-mutagenic metabolite in Salmonella typhimurium

Salmonella typhimurium strains (OASS-positive) synthesize a toxic but non-mutagenic metabolite from cyanide and O-acetylserine. Salmonella typhimurium mutant DW379 (OASS-deficient) is neither able to carry out this reaction in vitro nor produce the toxic metabolite in vivo. L-Cysteine reverses the cyanide metabolite mediated inhibition and thus allows OASS-positive strains to grow in medium containing cyanide and O-acetylserine. The results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of cyanide and O-acetylserine to form the toxic metabolite. This metabolite is ninhydrin-positive, adheres strongly to the cation-exchange column, and migrates in TLC to an Rf value similar to that of beta-cyanoalanine.     

O2-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae.

Mutant strains of the yeast Saccharomyces cerevisiae which lack functional Cu,Zn superoxide dismutase (SOD-1) do not grow aerobically unless supplemented with methionine. The molecular basis of this O2-dependent auxotrophy in one of the mutants, Dscd1-1C, has been investigated. Sulfate supported anaerobic but not aerobic mutant growth. On the other hand, cysteine and homocysteine supported aerobic growth while serine, O-acetylserine, and homoserine did not, indicating that the interconversion of cysteine and methionine (and homocysteine) was not impaired. Thiosulfate (S2O3(2-] and sulfide (S2-) also supported aerobic growth; the activities of thiosulfate reductase and sulfhydrylase in the aerobic mutant strain were at wild-type levels. Although the levels of SO4(2-) and adenosine-5'-sulfate (the first intermediate in the SO4(2-) assimilation pathway) were elevated in the aerobically incubated mutant strain, this condition could be attributed to a decrease in protein synthesis caused by the de facto sulfur starvation and not to a block in the pathway. Therefore, the activation of SO4(2-) (to form 3'-phosphoadenosine-5'-phosphosulfate) appeared to be O2 tolerant. Sulfite reductase activity and substrate concentrations [( NADPH] and [SO3(2-)]) were not significantly different in aerobically grown mutant cultures and anaerobic cultures, indicating that SOD-1- mutant strains could reductively assimilate sulfur oxides. However, the mutant strain exhibited an O2-dependent sensitivity to SO3(2-) concentrations of less than 50 microM not exhibited by any SOD-1+ strain or by SOD-1- strains supplemented with a cytosolic O2(-)-scavenging activity. This result suggests that the aerobic reductive assimilation of SO4(2-) at the level of SO3(2-) may generate a cytotoxic compound(s) which persists in SOD-(1-) yeast strains.     

Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity.

Summary Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A⁺ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23°C and after shift to 37°C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.