JOURNAL OF BACTERIOLOGY, Aug. 1987, p. 3458-3463 Vol. 169, No. 8 0021-9193/87/083458-06$02.00/0 Copyright X) 1987, American Society for Microbiology Partial Purification and Some Properties of Homoserine O-Acetyltransferase of a Methionine Auxotroph of Saccharomyces cerevisiae SHUZO YAMAGATA Department ofBiology, Faculty of General Education, Gifu University, Gifu 501-11, Japan Received 15 December 1986/Accepted 27 April 1987 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, Phenyl- Sepharose 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 the acetyl exchange reaction (equation 2) in a crude extract. few enteric bacteria O-acetyl-L-homoserine is an essential However, purification and further characterization of the member of the biosynthetic pathway from L-homoserine to enzyme fromn this organism have not yet been described, L-methionine (9, 23). In many cases, O-acetylhomoserine mainly because the amount of the protein in the cell is very synthesizes cystathionine with L-cysteine through the small and the protein is also very unstable after extraction. cystathionine y-synthase reaction. Cystathionine is then To characterize the enzyme of S. cerevisiae, strains with cleaved through the .-cystathionase reaction to produce high enzyme levels and a stabilization method were sought. L-homocysteine. Butin some microorganisms, O-acetylhomo- In this paper, both partial purification of the enzyme from serine can also be sulfhydrylated with H2S through catalysis a methionine auxotroph of S. cerevisiae and its enzymatic by O-acetylhomnoserine sulfhydrylase, giving rise to L- properties are dealt with. homocysteine directly (7, 17, 19, 20, 30, 31). The enzyme homoserine O-acetyltransferase (EC 2.3. MATERIALS AND METHODS 1.31), which catalyzes transfer of the acetyl group from Organisms. Methionine-requiring mutant strains of the acetyl coenzyme A (acetyl-CoA) to the 0 atom of yeast S. cerevisiae were isolated by Naiki and Iwata (18) homoserine (see equation 1 below), also catalyzes the acetyl from a haploid strain (a mating type, galactose fermentable, exchange reaction between O-acetylhomoserine and pantothenate nonrequiring). The strains employed for inves- [14C]homoserine (see equation 2) (9, 23). tigating synthesis of the enzyme are referred to in this paper L-Homoserine + acetyl-CoA -+ O-acetyl-L-homoserine as the wild-type strain and six mutant strains derived from it; + CoA (1) two require sulfite (strains 12 and 15), two require thiosulfate (strains 6 and 8), and two require cysteine (strains 13 and 17). O-Acetyl-L-homoserine + L-[14C]homoserine a± Chemicals and other materials. Synthesis of O-acetyl-L- L-homoserine + O-acetyl-L-[14C]homoserine (2) amino acids (21) and purification of O-acetylserine-O-acetyl- homoserine sulfhydrylase of S. cerevisiae (29) were carried This enzyme has not yet been well characterized except for out as described elsewhere. DEAE-cellulose (DE-52; a few bacteria (16, 22, 26, 27). Shiio and colleagues (16, 22) Whatman), Sephadex G-150 (Pharmacia Fine Chemicals), have recently reported on feedback control by the end Phenyl-Sepharose C1-4B (Pharmacia), and carrier ampholite product methionine in the case of the enzyme from (pH 3.5 to 5.0; LKB Produkter) were obtained commer- Brevibacterium flavum. Wyman et al. have described puri- cially. Bovine liver catalase, acetyl-CoA, and yeast alcohol fication of the enzyme to near homogeneity (26) and also its dehydrogenase were products of Boehringer Mannheim. regulation in whole cells (27) of Bacillus polymyxa. This Rabbit muscle lactate dehydrogenase, bovine serum albu- protein is, however, subject to rapid and irreversible inacti- min, succinyl coenzyme A (succinyl-CoA), and O-succinyl- vation after extraction (26). L-homoserine were products of Sigma Chemical Co. Mutant Regulatory properties of the enzyme of Saccharomyces strains of S. cerevisiae were gifts from N. Naiki. cerevisiae have been reported (4, 6), based on observation of Culture of cells for analysis of enzyme synthesis. Cells of 3458 VOL. 169, 1987 HOMOSERINE O-ACETYLTRANSFERASE IN S. CEREVISIAE 3459 each strain were cultured in a synthetic medium (18) supple- TABLE 1. Comparison of amounts of homoserine 0- mented with 0.1 mM L-cysteine or L-methionine. One 3-liter acetyltransferase in methionine auxotrophs of S. cerevisiae flask containing 1.5 liters of the medium was inoculated with Homoserine acetyltransferase activity 20 ml of a fully-grown seed culture of a given strain and then Strain mU/mg of protein Total UG shaken vigorously at 30°C. The cells were harvested in early stationary phase by centrifugation at 3,000 x g for 5 min and Wild type 0 0 subsequently washed with 500 ml of cold distilled water. 6 24.7 3.80 Extraction. Cell pellets (about 10 g from 3 liters of culture 8 1.52 0.28 were in cold buffer A mM Tris 12 20.3 3.31 medium) suspended (50 13 0 0 hydrochloride buffer, pH 7.8, containing 25% sucrose, 10 15 0 0 mM DL-homoserine, and 2.5 mM dithiothreitol). After the 17 7.22 0.43 volume was adjusted to 40 ml, the suspensions were agitated a in a Dyno Mill (W. A. Bachofen Maschinenfabrik, type Amount of enzyme contained in cells cultured in a total volume of 3 liters of a synthetic medium (18) supplemented with 0.1 mM L-cysteine hydrochlo- KDL) with 65 ml ofglass beads (0.25 to 0.50 mm in diameter) ride. at 3,000 rpm for two 90-s periods with an interval of 60 s. The homogenate was centrifuged at 10,000 x g for 30 min. The supernatant solution obtained was concentrated with ml of the equilibration buffer, together with 0.1 mg of bovine Aquacide II-A (Calbiochem) overnight and then dialyzed liver catalase (EC 1.11.1.6) (molecular weight [MW], against 350 ml of buffer A overnight. The dialyzed materials 248,000), 0.5 mg of yeast alcohol dehydrogenase (EC 1.1.1.1) (approximately 5 ml) were analyzed for homoserine 0- (MW 150,000), 0.5 mg of rabbit muscle lactate dehydrogen- acetyltransferase activity. ase (EC 1.1.1.27) (MW 140,000), and 5 mg of bovine serum Assay of homoserine O-acetyltransferase activity. The reac- albumin (MW 68,000), and then applied to the column. The tion mixture contained, in a total volume of 1.5 ml, 100 mM eluate was fractionated by 1.1-ml fractions. MW was calcu- potassium phosphate buffer (pH 7.5), 0.65 mM 5,5'- lated by the method of Whitaker (25). dithiobis(2-nitrobenzoic acid), 0.13 mM acetyl-CoA, 25% MW was also determined by centrifugation of the enzyme sucrose, 10 mM L-homoserine, and an appropriate amount of in a sucrose concentration gradient. Twenty-six milliunits of enzyme. The reaction was carried out at 30°C. Change in the enzyme were dissolved together with 1 mg of rabbit liver absorbance at 412 nm was recorded with and without lactate dehydrogenase in 1 ml of 50 mM Tris hydrochloride homoserine. Consumption of acetyl-CoA during the homo- buffer, pH 7.8, containing 1 mM EDTA and 0.2 mM dithio- serine acetyltransferase reaction was calculated from the threitol. The solution was layered over a sucrose concentra- difference between the two changes in absorbance of re- tion gradient (5 to 30%) formed in the same buffer and duced 5,5'-dithiobis(2-nitrobenzoic acid), using an EmM at 1 subsequently centrifuged at 2°C and 40,000 rpm for 25 h. The cm of 13.6 (8) (assay I). Decreases in the acetyl-CoA MW was calculated by the method of Martin and Ames (15). concentration in the reaction mixture were also determined Other procedures. Electrofocusing was carried out at in the absence of 5,5'-dithiobis(2-nitrobenzoic acid) by ob- 0.5°C by the method of Vesterberg and Svensson (24), with serving the decrease in absorbance at 232 nm. The change in carrier ampholite (pH 3.5 to 5.0) at an average concentration concentration was calculated with an EmM at 1 cm of 4.5 for of 1%. To protect the enzyme from inactivation, sucrose was the thioether bond (1) (assay II). Assay I was always used added at an average concentration of 38%.