O-acetylhomoserine Sulfhydrylase Research Articles

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

PH-dependent regulation of an acidophilic O-acetylhomoserine sulfhydrylase from Lactobacillus plantarum.

In the present study, we can elucidate the pH-dependent regulation mechanism of the acidophilic OAHS. The acidophilic feature of the enzyme is caused by the introduction of an acidic residue to the neighborhood of the key acidic residue acting as a switch for the structural interconversion. The strategy may be useful in the field of protein engineering to change the optimal pH of the enzymes. In addition, this study may be useful for the development of antibacterial drugs because the l-methionine synthesis essential for bacteria is inhibited by the OAHS inhibitors. The compounds that can inhibit the interconversion between the open and closed forms of OAHS may become antibacterial drugs.

O-acetyl-homoserine sulfhydrylase deficient Streptococcus suis serotype 2 strain SC19 becomes an avirulent strain and provides immune protection against homotype infection in mice

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5’-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host’s BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.

О-Аcetylhomoserine sulfhydrylase from <i>Clostridioides difficile</i>: the role of tyrosine residues of the active center

O-acetylhomoserine sulfhydrylase is one of the key enzymes in the biosynthesis of methionine in Clostridioides difficile. The mechanism of the γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among pyridoxal-5′-phosphate-dependent enzymes involved in the metabolism of cysteine and methionine. To clarify the role of the active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements for phenylalanine and alanine were obtained. The catalytic and spectral properties of mutant forms were investigated. The rate of the γ-substitution reaction catalyzed by mutant forms with the replacement of the Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the residues Tyr52 and Tyr107 led to a decrease in the affinity of the apoenzyme to the coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring the optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 can act as a general acid catalyst at the stage of acetate elimination.

Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae

Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S. cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation.

O-acetylhomoserine sulfhydrylase from Clostridium novyi. Cloning, expression of the gene and characterization of the enzyme

The gene NT01CX_1210 of pathogenic bacterium Clostridium novyi annotated as encoding O-acetylhomoserine sulfhydrylase was cloned and expressed in Escherichia coli. The gene product having O-acetylhomoserine sulfhydrylase activity was purified to homogeneity. The protein showed molecular mass of approximately 184 kDa for the native form and 46 kDa for the subunit. The enzyme catalyzes the γ-substitution reaction of O-acetylhomoserine with maximum activity at pH 7.5. Analysis of C. novyi genome allowed us to suggest that there is only one way for the synthesis of l-methionine in the bacterium. The data obtained may provide the basis for further study of the role of OAHS in Clostridium bacteria and an ascertainment of its mechanism.

Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

Novel biosynthetic pathway for sulfur amino acids in Cryptococcus neoformans.

We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met- mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide.

The GLO1 Gene Is Required for Full Activity of O-Acetyl Homoserine Sulfhydrylase Encoded by MET17.

During glycolysis, yeast generates methylglyoxal (MG), a toxic metabolite that affects growth. Detoxification can occur when glyoxylase I (GLO1) and glyoxylase II (GLO2) convert MG to lactic acid. We have identified an additional, previously unrecognized role for GLO1 in sulfur assimilation in the yeast Saccharomyces cerevisiae. During a screening for putative carbon-sulfur lyases, the glo1 deletion strain showed significant production of H2S during fermentation. The glo1 strain also assimilated sulfate inefficiently but grew normally on cysteine. These phenotypes are consistent with reduced activity of the O-acetyl homoserine sulfhydrylase, Met17p. Overexpression of Glo1p gave a dominant negative phenotype that mimicked the glo1 and met17 deletion strain phenotypes. Western analysis revealed reduced expression of Met17p in the glo1 deletion, but there was no indication of an altered conformation of Met17p or any direct interaction between the two proteins. Unravelling a novel function in sulfur assimilation and H2S generation in yeast for a gene never connected with this pathway provides new opportunities for the study of this molecule in cell signaling, as well as the potential regulation of its accumulation in the wine and beer industry.

Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.

BackgroundOver the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains.ResultsWe compared titer and quality of several Nanobodies® produced in wild type and MutS strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in MutS strains contained a product variant with a Δ−16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-l-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium.ConclusionWe showed that misincorporation of methoxine occurs particularly in P. pastoris MutS strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0499-2) contains supplementary material, which is available to authorized users.

Metaproteomics reveals the major microbial players and their biogeochemical functions in a productive coastal system in the northern South China Sea.

We study the metaproteome of the GF/F-prefiltered fraction of a microbial community from Shantou coast summer surface waters using a shotgun proteomic approach. Spectra attributed to the marine Roseobacter clade (MRC), the oligotrophic marine Gammaproteobacteria (OMG) group and Flavobacteria dominated in the microbial community, accounting for 21.0%, 23.2% and 12.7% of all of the detected spectra, respectively, whereas the SAR 92 clade accounted for 50% of the OMG group. The abundance of TonB-dependent receptors (TBDRs) was detected and the majority of TBDRs were attributed to the OMG, whereas a large number of ABC transporters matched to the MRC, which suggests niche separation in the microbial community. Expression of proteorhodopsin and RagB/SusD from Flavobacteria facilitates their attachment and growth on algal-derived organic matter. Taurine and glycine betaine appear to be an important source of carbon and nitrogen for the Rhodobacteraceae and SAR11 cluster. The detection of carbon monoxide dehydrogenase, formate dehydrogenase, O-acetylhomoserine sulfhydrylase and sulfur oxidation protein from the MRC demonstrated that members of the MRC play important roles in coastal ocean biogeochemical cycles. This study provides the first insight into functional processes occurring in microbial communities in coastal waters in the South China Sea.

Coupling Bioorthogonal Chemistries with Artificial Metabolism: Intracellular Biosynthesis of Azidohomoalanine and Its Incorporation into Recombinant Proteins

In this paper, we present a novel, “single experiment” methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of l-azidohomoalanine from O-acetyl-l-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph E. coli strain. In our system, the host’s methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient l-azidohomoalanine incorporation in place of l-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.

A novel mechanism of sulfur transfer catalyzed by O-acetylhomoserine sulfhydrylase in the methionine-biosynthetic pathway of Wolinella succinogenes.

O-Acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate (PLP) dependent sulfide-utilizing enzyme in the L-cysteine and L-methionine biosynthetic pathways of various enteric bacteria and fungi. OAHS catalyzes the conversion ofO-acetylhomoserine to homocysteine using sulfide in a process known as direct sulfhydrylation. However, the source of the sulfur has not been identified and no structures of OAHS have been reported in the literature. Here, the crystal structure of Wolinella succinogenes OAHS (MetY) determined at 2.2 Å resolution is reported. MetY crystallized in space group C2 with two monomers in the asymmetric unit. Size-exclusion chromatography, dynamic light scattering and crystal packing indicate that the biological unit is a tetramer insolution. This is further supported by the crystal structure, inwhich a tetramer is formed using a combination of noncrystallographic and crystallographic twofold axes. A search for structurally homologous proteins revealed that MetY has the same fold as cystathionine γ-lyase and methionine γ-lyase. The active sites of these enzymes, which are also PLP-dependent, share a high degree of structural similarity, suggesting that MetY belongs to the γ-elimination subclass of the Cys/Met metabolism PLP-dependent family of enzymes. The structure of MetY, together with biochemical data, provides insight into the mechanism of sulfur transfer to a small molecule via a protein thiocarboxylate intermediate.

Expression, purification and preliminary crystallographic analysis of O-acetylhomoserine sulfhydrylase from Mycobacterium tuberculosis.

The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1 Å.

Towards methionine overproduction in Corynebacterium glutamicum - methanethiol and dimethyldisulfide as reduced sulfur sources

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur source for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis has predicted a high methionine yield for these reduced compounds provided that they can be utilized. Wild type cells were able to grow on methanethiol and on dimethyldisulfide as sole sulfur source, respectively. Isotope labeling studies with mutant strains exhibiting targeted modification of methionine biosynthesis gave detailed insight into the underlying pathways involved in assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as entire molecule, adding the terminal S-CH3 group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as enzyme catalyzing this reaction. Deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur source. Plasmid based overexpression of metY in the delta metY background restored the capability to grow on methanethiol or dimethyldisulfide as sole sulfur source. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased two-fold in the engineered strain. This positive effect was limited by depletion of the metY substrate O-acetylhomoserine, requesting for further metabolic engineering targets towards competitive production strains.

The cmaR gene of Corynebacterium ammoniagenes performs a novel regulatory role in the metabolism of sulfur-containing amino acids

A novel regulatory gene, which performs an essential function in sulfur metabolism, has been identified in Corynebacterium ammoniagenes and was designated cmaR (cysteine and methionine regulator in C. ammoniagenes). The cmaR-disrupted strain (DeltacmaR) lost the ability to grow on minimal medium, and was identified as a methionine and cysteine double auxotroph. The mutant strain proved unable to convert cysteine to methionine (and vice versa), and lost the ability to assimilate and reduce sulfate to sulfide. In the DeltacmaR strain, the mRNAs of the methionine biosynthetic genes metYX, metB and metFE were significantly reduced, and the activities of the methionine biosynthetic enzymes cystathionine gamma-synthase, O-acetylhomoserine sulfhydrylase, and cystathionine beta-lyase were relatively low, thereby suggesting that the cmaR gene exerts a positive regulatory effect on methionine biosynthetic genes. In addition, with the exception of cysK, reduced transcription levels of the sulfur-assimilatory genes cysIXYZ and cysHDN were noted in the cmaR-disrupted strain, which suggests that sulfur assimilation is also under the positive control of the cmaR gene. Furthermore, the expression of the cmaR gene itself was strongly induced via the addition of cysteine or methionine alone, but not the introduction of both amino acids together to the growth medium. In addition, the expression of the cmaR gene was enhanced in an mcbR-disrupted strain, which suggests that cmaR is under the negative control of McbR, which has been identified as a global regulator of sulfur metabolism. DNA binding of the purified CmaR protein to the promoter region of its target genes could be demonstrated in vitro. No metabolite effector was required for the protein to bind DNA. These results demonstrated that the cmaR gene of C. ammoniagenes plays a role similar to but distinct from that of the functional homologue cysR of Corynebacterium glutamicum.

Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress

The cysteine biosynthesis pathway differs between plants and the yeast Saccharomyces cerevisiae. The yeast MET25 gene encoded to O-acetylhomoserine sulfhydrylase (AHS) catalyzed the reaction that form homocysteine, which later can be converted into cystiene. In vitro studies show that this enzyme possesses also the activity of O-acetyl(thiol)lyase (OASTL) that catalyzes synthesis of cysteine in plants. In this study, we generated transgenic tobacco plants expressing the yeast MET25 gene under the control of a constitutive promoter and targeted the yeast protein to the cytosol or to the chloroplasts. Both sets of transgenic plants were taller and greener than wild-type plants. Addition of SO(2), the substrate of the yeast enzyme caused a significant elevation of the glutathione content in representative plants from each of the two sets of transgenic plants expressing the yeast gene. Determination of non-protein thiol content indicated up to four-folds higher cysteine and 2.5-fold glutathione levels in these plants. In addition, the leaf discs of the transgenic plants were more tolerant to toxic levels of sulphite, and to paraquat, an herbicide generating active oxygen species.

Cloning of the O -Acetylhomoserine Sulfhydrylase Gene from the Ruminal Bacterium Selenomonas ruminantium HD4

The O-acetylhomoserine sulfhydrylase (OAHS) gene was cloned from a Selenomonas ruminantium HD4 Lambda ZAP II genomic library by degenerative probe hybridization and complementation. Sequence analysis revealed an 869-bp ORF with a G + C content of 53%. The ORF had significant homology with enzymes involved in homocysteine biosynthesis. A CuraBLASTN homology search showed that the ORF has 63% nucleotide identity with the OAHS of Bacillus stearothermophilus, Corynebacterium glutamicum, and Acremonium chrysogenum, and has 58% identity with met25 of Saccharomyces cerevisiae and metZ of Pseudomonas aeruginosa. The deduced amino acid sequence exhibited 45% similarity with Met25 and MetZ. Further analysis predicted that the gene product was a member of the pyridoxal phosphate enzyme family. Complementation experiments with Escherichia coli metA, metB, and metC mutant strains showed that the S. ruminantium OAHS gene can complement the metC mutation and allow for growth on minimal media that contained sodium thiosulfate as the sole source of sulfur. When the OAHS was disturbed by inserting a EZ::TN pMOD-2<Apramycin> transposon, the complementation was lost. Therefore, these results suggest that the gene functions as OAHS in S. ruminantium HD4.

Homocysteine biosynthesis pathways of Streptococcus anginosus

A gene ( cgs) encoding cystathionine γ-synthase was cloned from Streptococcus anginosus, and its protein was purified and characterized. The cgs gene and the immediately downstream lcd gene were shown to be cotranscribed as an operon. High-performance liquid chromatography analyses showed that the S. anginosus Cgs not only has cystathionine γ-synthase activity, but also expresses O-acetylhomoserine sulfhydrylase activity. These results suggest that S. anginosus has the capacity to utilize both the transsulfuration and direct sulfhydrylation pathways for homocysteine biosynthesis.

Corynebacterium glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis.

A direct sulfhydrylation pathway for methionine biosynthesis in Corynebacterium glutamicum was found. The pathway was catalyzed by metY encoding O-acetylhomoserine sulfhydrylase. The gene metY, located immediately upstream of metA, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 Da. In accordance with DNA and protein sequence data, the introduction of metY into C. glutamicum resulted in the accumulation of a 47-kDa protein in the cells and a 30-fold increase in O-acetylhomoserine sulfhydrylase activity, showing the efficient expression of the cloned gene. Although disruption of the metB gene, which encodes cystathionine gamma-synthase catalyzing the transsulfuration pathway of methionine biosynthesis, or the metY gene was not enough to lead to methionine auxotrophy, an additional mutation in the metY or the metB gene resulted in methionine auxotrophy. The growth pattern of the metY mutant strain was identical to that of the metB mutant strain, suggesting that both methionine biosynthetic pathways function equally well. In addition, an Escherichia coli metB mutant could be complemented by transformation of the strain with a DNA fragment carrying corynebacterial metY and metA genes. These data clearly show that C. glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. Although metY and metA are in close proximity to one another, separated by 143 bp on the chromosome, deletion analysis suggests that they are expressed independently. As with metA, methionine could also repress the expression of metY. The repression was also observed with metB, but the degree of repression was more severe with metY, which shows almost complete repression at 0.5 mM methionine in minimal medium. The data suggest a physiologically distinctive role of the direct sulfhydrylation pathway in C. glutamicum.