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

• Published in: Journal of Bacteriology Vol. 175; no. 15; pp. 4800 - 4808
• Main Authors: Yamagata, S, D'Andrea, R.J, Fujisaki, S, Isaji, M, Nakamura, K
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-08-1993
• Subjects: Acetyltransferases - analysis; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Base Sequence; Biological and medical sciences; Cloning, Molecular; Cystathionine gamma-Lyase - chemistry; Cystathionine gamma-Lyase - genetics; Cystathionine gamma-Lyase - metabolism; ESCHERICHIA; Escherichia coli; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; GENE; Gene Expression - genetics; GENES; Genes, Fungal - genetics; Genes. Genome; Kinetics; LIASAS; LYASE; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation - genetics; PROPIEDADES FISICO-QUIMICAS; PROPRIETE PHYSICOCHIMIQUE; PURIFICACION; PURIFICATION; Recombinant Proteins - metabolism; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; SECUENCIA NUCLEICA; Sequence Homology; SEQUENCE NUCLEIQUE; Serine O-Acetyltransferase; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Cystathionine γ-lyase; Heterologous system; Yeast; Purification; Enzyme; Escherichia coli; Lyases; Gene expression; Fungi; Enzymatic activity; Gene; Carbon-sulfur lyases; Bacteria; Ascomycetes; Molecular cloning; Saccharomyces cerevisiae; Physicochemical properties; Enterobacteriaceae; Thallophyta
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/jb.175.15.4800-4808.1993

By screening a yeast genomic library, we isolated and characterized a gene rescuing the cysteine requirement in a "cys1" strain of Saccharomyces cerevisiae. Except for four residues in the open reading frame composed of 1,182 nucleotides, the DNA sequence was the same as that for the CYS3 (CYI1) 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. Oshima, and A. Toh-e, J. Bacteriol. 174:pp.3339-3347, 1992). S. cerevisiae "cys1" strains carry two closely linked mutations; one (cys1) causes 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-lyase is consistent with both defects being responsible for the cysteine auxotrophy. In an effort to further determine the physicochemical and enzymatic properties of this enzyme, a coding fragment was cloned into an Escherichia coli expression plasmid, and the protein was produced in the bacteria. The induced protein was extracted by sonication and purified to homogeneity through one course of DEAE-cellulose column chromatography. The yield of the protein was approximately 150 mg from cells cultured in 1 liter of L broth. The protein showed molecular weights of approximately 194,000 and 48,000 (for the subunit), suggesting a tetrameric structure. An s20,w value of 8.8 was estimated by centrification in a sucrose concentration gradient. No sulfhydryl groups were detected, which is consistent with the absence of cysteine residues in the coding sequence. The isoelectric point was at pH 5.2. The protein showed a number of cystathionine-related activities, i.e., cystathionine beta-lyase (EC 4.4.1.8), cystathionine gamma-lyase, and cystathionine gamma-synthase (EC 4.2.99.9) with L-homoserine as a substrate. In addition, we demonstrated L-homoserine sul