Partial purification and some properties of homoserine O-acetyltransferase of a methionine auxotroph of Saccharomyces cerevisiae

Abstract

A wild-type strain and six methionine auxotrophs of Saccharomyces cerevisiae were cultured in a synthetic medium supplemented with 0.1 mM L-cysteine or L-methionine and analyzed for the synthesis of homoserine O-acetyltransferase (EC 2.3.1.31). Among them, four muitant strains exhibited enzyme activity in cell extracts, Methionine added to the synthetic medium at concentrations higher than 0.1 mM repressed enzyme synthesis in two of these strains. The enzyme was partially purified (3,500-fold) from an extract of a mutant strkin through ammonium sulfate fractionation and chromatography on columns of DEAE-cellulose, PhenylSepharose C1-4B, and Sephadex G-150. The enzyme exhibited optimal p1I at 7.5 for activity and at 7.8 for stability. The reaction product was ascertained to be O-acetyl-L-homoserine by confirming that it produced L-homocysteine in an O-acetyl-L-homoserine sulfhydrylase reaction. The Km for L-homoserine was 1.0 mM, and for acetyl coenzyme A it was 0.027 mM. The molecular weight of the enzyme was esimated to be approximately 104,000 by Sephadex G-150 column chromatography anid 101,000 by sucrose density gradient centrifugation.The isoelectric point was at pH 4.0. Of the hydroxy amino acids examined, the enzyme showed reactivity only to L-homoserine. Succinyl coenzyme A was not an acyl donor. In the absence of L-homoserine, acetyl coenzyme A was deacylated by the enzyme, with a Km of 0.012 mM. S-Adenosylmethionine and S-adenosylhomocysteine slightly inhibited the enzyme, but nmethionine had no effect.It is well established that in microorganisms other than a few enteric bacteria O-acetyl-L-homoserine is an essential member of the biosynthetic pathway from L-homoserine to L-methionine (9, 23). In many cases, O-acetylhomoserine synthesizes cystathionine with L-cysteine through the cystathionine y-synthase reaction. Cystathionine is then cleaved through the .-cystathionase reaction to produce L-homocysteine. Butin some microorganisms, O-acetylhomoserine can also be sulfhydrylated with H2S through catalysis by O-acetylhomnoserine sulfhydrylase, giving rise to Lhomocysteine directly (7,17,19,20,30,31).The enzyme homoserine O-acetyltransferase (EC 2.3. 1.31), which catalyzes transfer of the acetyl group from acetyl coenzyme A (acetyl-CoA) to the 0 atom of homoserine (see equation 1 below), also catalyzes the acetyl exchange reaction between O-acetylhomoserine and [14C]homoserine (see equation 2) (9, 23).(2) This enzyme has not yet been well characterized except for a few bacteria (16,22,26,27). Shiio and colleagues (16,22) have recently reported on feedback control by the end product methionine in the case of the enzyme from Brevibacterium flavum. Wyman et al. have described purification of the enzyme to near homogeneity (26) and also its regulation in whole cells (27) of Bacillus polymyxa. This protein is, however, subject to rapid and irreversible inactivation after extraction (26).Regulatory properties of the enzyme of Saccharomyces cerevisiae have been reported (4, 6), based on observation of the acety...

The role of amino acids in the regulation of hydrogen sulfide production during ultradian respiratory oscillation of Saccharomyces cerevisiae

“…4b) concentrations decreased H 2 S production immediately, and increased respiration for 14 min. These results were in accordance with the prevailing view that only homoserine, not serine, is converted to O-acetylhomoserine by homoserine O-acetyltransferase in S. cerevisiae (Yamagata 1987;Cherest and Surdin-Kerjan 1992).…”

“…The supernatant obtained was concentrated by freeze-drying for 40 h, dissolved with 5 ml of distilled water, dialyzed against 4 l of 5 mM Tris buffer (pH 7.6), and then analyzed for enzyme activity. Serine O-acetyltransferase and homoserine O-acetyltransferase were assayed at 30°C by measuring the amount of CoA produced (Kredich and Becker 1971;Yamagata 1987). In the case of homoserine O-acetyltransferase, L-homoserine was substituted for L-serine.…”

“…sulfide, methionine, cysteine, S-adenosylmethionine and glutathione). However, the mechanisms of regulation of sulfate assimilation are still a matter of controversy, especially regarding O-acetylserine synthesis and the role of O-acetylhomoserine/O-acetylserine sulfhydrylase, cysteine, methionine and S-adenosylmethionine in the SA pathway (Ono et al 1991(Ono et al , 1996Yamagata et al 1974Yamagata et al , 1975Yamagata et al , 1994Yamagata 1987;Cherest and Surdin-Kerjan 1992;Cherest et al 1993).…”

Cloning and bacterial expression of the CYS3 gene encoding cystathionine gamma-lyase of Saccharomyces cerevisiae and the physicochemical and enzymatic properties of the protein

“…Reactions were carried out in 0.5 ml of mixtures containing 100 mM potassium phosphate buffer (pH 8.0), 5 mM CTIT, 0.2 mM PLP, and 0.250 mg of the protein for the indicated time. After stopping the reactions with 0.05 ml of 30% trichloroacetic acid, the reaction mixture was treated through a small column of Dowex 5OW-X8, and the products were analyzed by high-voltage paper electrophoresis after oxidation with performic acid as described previously (48). Other conditions are described in the legend to Fig.…”

“…Gel filtration was carried out at 4°C on a Sephacryl S-300 HR column (1.5 by 64 cm) equilibrated with 50 mM Tris-hydrochloride (or 100 mM potassium phosphate) buffer (pH 7.8) containing 1 mM EDTA, 100 mM NaCl, 0.2 mM PLP, and 0.05 mM dithiothreitol. Chromatography of the gene product (0.7 mg of protein) and standard proteins was carried out as described previously (48). Gel filtration was also carried out at room temperature by high-pressure liquid chromatography on a TSK gel G3000SW column (0.75 by 30 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.0) containing 200 mM potassium sulfate.…”