CLONING AND BACTERIAL EXPRESSION OF THE CYS3 GENE ENCODING CYSTATHIONINE GAMMA-LYASE OF SACCHAROMYCES-CEREVISIAE AND THE PHYSICOCHEMICAL AND ENZYMATIC-PROPERTIES OF THE PROTEIN

By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a ''cys1'' strain of Sacchar omyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that f or the CYS3 (CY11) gene, encoding cystathionine gamma-lyase (EC 4.4.1. 1), and isolated previously as a cycloheximide-induced gene (B. Ono, K . Tanaka, K. Naito, C. Heike, S. Shinoda, S. Yamamoto, S. Ohmori, T. O shima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevis iae ''cys1'' strains carry two closely linked mutations; one (cys1) ca uses a defect in serine O-acetyltransferase (EC 2.3.1.30), and another , designated cys3, impairs cystathionine gamma-lyase activity. Rescue of the cysteine requirement by the gene encoding cystathionine gamma-l yase is consistent with both defects being responsible for the cystein e auxotrophy. In an effort to further determine the physicochemical an d enzymatic properties of this enzyme, a coding fragment was cloned in to an Escherichia coli expression plasmid, and the protein was produce d in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column c hromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular wei ghts of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centri fugation in a sucrose concentration gradient. No sulfhydryl groups wer e detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The prote in showed a number of cystathionine-related activities, i.e., cystathi onine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathi onine gamma-synthase (EC 4.2.99.9) with L-homoserine as a substrate. I n addition, we demonstrated L-homoserine sulfhydrylase (adding H2S) ac tivity but could find no detectable serine O-acetyltransferase activit y. In this paper, we compare the enzymatic properties of the protein w ith those of homologous enzymes previously reported and discuss the po ssibility that this enzyme has a physiological role as cystathionine b eta-lyase and cystathionine gamma-synthase in addition to its previous ly described role as cystathionine gamma-lyase.