Shuzo Yamagata

Roles of O-acetyl-l-homoserine sulfhydrylases in microorganisms

O-Acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) is essential for certain micro-organisms, functioning as a homocysteine synthase in the pathway of methionine synthesis. It participates in an alternative pathway of L-homocysteine synthesis for those microbes in which homocysteine is synthesized mainly via cystathionine. The protein can also catalyze the de novo synthesis of L-cysteine and O-alkyl-L-homoserine in some microorganisms. The enzyme possibly recycles the methylthio group of methionine.

Bacillus okuhidensis sp. nov., isolated from the Okuhida spa area of Japan.

Two Gram-positive, endospore-forming, alkaliphilic bacteria were isolated from water samples obtained from the Okuhida hot spa area of Japan. The unknown bacteria were characterized using phenotypic and molecular taxonomic methods. On the basis of phylogenetic evidence and phenotypic distinctiveness, a new species, Bacillus okuhidensis sp. nov., is proposed. The type strain of Bacillus okuhidensis is GTC 854T (= JCM 10945T = DSM 13666T).

Differential Recognition of Citrate and a Metal-Citrate Complex by the Bacterial Chemoreceptor Tcp

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the α1-α2 and α3-α4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn67, which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn67 and allosteric switching.

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.

Mutational analysis of ligand recognition by tcp, the citrate chemoreceptor of Salmonella enterica serovar typhimurium.

The chemoreceptor Tcp mediates taxis to citrate. To identify citrate-binding residues, we substituted cysteine for seven basic or polar residues that are chosen based on the comparison of Tcp with the well-characterized chemoreceptors. The results suggest that Arg-63, Arg-68, Arg-72, Lys-75, and Tyr-150 (and probably other unidentified residues) are involved in the recognition of citrate.

Overexpression of the Saccharomyces cerevisiae MET17/MET25 gene in Escherichia coli and comparative characterization of the product with O-acetylserine O-acetylhomoserine sulfhydrylase of the yeast

The Saccharomyces cerevisiae MET17/MET25 gene encoding O-acetyl-L-serine (OAS)·O-acetyl-L-homoserine (OAH) sulfhydrylase (EC 4.2.99.10) was overexpressed in Escherichia coli and the gene product was purified to homogeneity, using three steps, with a recovery of 28% from the total cell extract. The gene product has been compared with OAS·OAH sulfhydrylase purified from the yeast cells. These two protein preparations were indistinguishable with respect to their behavior in polyacrylamide gel electrophoresis, both with and without sodium dodecyl sulfate, their specificity for substrate amino acids, Michaelis constant (Km) value for OAH, sensitivity to carbonyl reagents, absorption spectrum, isoelectric point, behavior in HPLC (both ion-exchange chromatography and gel filtration), sensitivity to heat treatment, susceptibility to trypsin digestion, and their N-terminal amino acid sequence. The results obtained imply that the gene product is properly processed in E. coli, and the technique developed in this study to overexpress the gene in bacterial cells provides us with a large amount of the purified preparation of the enzyme. In contrast to a previous report we found that cystathionine γ-lyase of S. cerevisiae behaved differently from OAS·OAH sulfhydrylase during the purification procedure.

Cysteine Synthase of an Extremely Thermophilic Bacterium, Thermus thermophilus HB8

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70°C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80°C with 10 mM dithiothreitol. The enzyme was also quite stable at pH 8–12 (50°C, 30 min). It had an apparent K m of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a V max of 435 μmol/min/mg of protein. The apparent K m for sulfide was approximately 50 μM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5′-phosphate as a cofactor. The apo-enzyme showed an apparent K m of 29 μM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.

Characterization of O-Acetyl-L-serine Sulfhydrylase Purified from an Alkaliphilic Bacterium

O-Acetyl-L-serine sulfhydrylase (EC 4.2.99.8) activity was shown to be very high compared with O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity and L-cystathionine cleaving activities, in an extract of cells of an alkaliphilic bacterium grown in a synthetic medium. The synthesis of the first enzyme was repressed by approximately 55% by both L-cystine and L-djenkolic acid added to the medium at a concentration of 0.5 mM, but L-methionine (1 mM) and S-adenosyl-L-methionine (0.5 mM) affected it to lesser extents. Its enzyme activity was inhibited by 25% and 12% by methionine (10 mM) and S-adenosylmethionine (5 mM), respectively. The enzyme was purified from the extract through ammonium sulfate fractionation, heat treatment, and chromatography on columns of DEAE-cellulose, Sephacryl S-300, and Octyl Sepharose CL-4B with a recovery of 21%. Polyacrylamide gel electrophoresis with sodium dodecylsulfate of the preparation obtained finally showed its homogeneity and the molecular mass of 37,000 Da for dissociated subunits. Gel filtration of the enzyme on a Sephacryl S-300 column showed an approximate molecular mass of 72,000 Da, suggesting that the enzyme was comprised of two identical subunits. The enzyme catalyzed the β-replacement reaction with O-acetylserine as a substrate, and showed no reactivity to other O-substituted amino acids tested. The reaction proceeded best at 40°C (when tested at pH 7.5), and at pH 6.5 (at 40°C). The enzyme kept 90% its activity after incubation at 65°C (at pH 7.5) for 30 min, and more than 90% after 30 min incubation at pHs 7-12 at 30°C. The enzyme had a K m of 4 mM for O-acetyl-L-serine and a V max of 37.0 μmol/min/mg of protein, a very low value compared with those of other organisms. However, the content of the enzyme in the extract was calculated to be approximately 3.5% total protein. Sensitivity of the enzyme to carbonyl reagents was very low, although it was shown to have pyridoxal 5′-phosphate as a cofactor by examination of its absorption spectrum. Sulfhydryl reagents tested showed no inhibition. The novelty of this enzyme among analogous sulfhydrylases purified from other organisms was discussed.

Immobilization of Saccharomyces cerevisiae Cystathionine γ-Lyase and Application of the Product to Cystathionine Synthesis

ABSTRACT Cystathionine γ-lyase of Saccharomyces cerevisiae was immobilized to aminohexyl-Sepharose through the cofactor pyridoxal 5′-phosphate and was characterized with respect to its cystathionine γ-synthase activity. The immobilized product was so stable that it repeatedly catalyzed as many as five cycles of the reaction without losing activity.

Substrate Inhibition of L-Cysteine α,β-Elimination Reaction Catalyzed by L-Cystathionine γ-Lyase of Saccharomyces cerevisiae

The α,β-elimination of L-cysteine catalyzed by Saccharomyces cerevisiae L-cystathionine γ-lyase (EC 4.4.1.1) was inhibited by the substrate. The absorption spectrum of the holoenzyme in the presence of L-cysteine showed that the substrate inhibition observed in this reaction was due mainly to removal of the cofactor.