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

Abstract

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 centrifugation 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 substrate. In addition, we demonstrated L-homoserine sulfhydrylase (adding H2S) activity but could find no detectable serine O-acteyltransferease activity. In this paper, we compare the enzymatic properties of the protein with those of homologous enzymes previously reported and discuss the possibility that this enzyme has a physiological role as cystathionine Beta-lyase and cystathionine gamma-synthase in addition to its previously described role as cystathionine gamma-lyase.

Substrate Inhibition ofL-Cysteine α,β-Elimination Reaction Catalyzed byL-Cystathionine γ-Lyase ofSaccharomyces cerevisiae

“…This is supported by the fact that CTT g-synthase reaction is catalyzed by this enzyme with cysteine as a substrate at high concentrations. [4][5][6]8) The increase in absorption at around 420 nm after the addition of cysteine was considered to be due to the increase in the external SchiŠ base with L-cysteine, since absorption at 418 nm is observed for Salmonella O-acetyl-L-serine sulfhydrylase binding its reaction product cysteine. 19) No absorption peak at 470-480 nm characteristic of an aminoacrylate intermediate reported for other amino acid-metabolizing enzymes 19,20) was observed for the enzyme with L-cysteine, although it showed absorption at the same wavelength with CTT and Lhomoserine (or its O-substituted derivatives) (data not shown).…”

“…The product (pyruvic acid) was mainly determined with lactic dehydrogenase (EC 1.1.1.27), 14) as described previously. 8) The color intensity of phenylhydrazone formed from 2,4-dinitrophenylhydrazine was assayed 15) in order to conˆrm the results obtained by using the former methods. 5,5?-Dithiobisnitrobenzoic acid was used to conˆrm the presence of a sulfhydryl group in the reaction products when Lcystine was used as the substrate.…”

“…The sample contained 0.3 ml of the holoenzyme solution and 0.9 ml of 0.1 M potassium phosphate buŠer (pH 7.8) containing 1 mM EDTA (ˆnal protein concentration, 8 mM enzyme, 32 mM subunit 8) ) and the spectrum (O) was determined against 1.2 ml of the same buŠer at 309 C. A solution (0.03 ml) of 100 mM Lcysteine hydrochloride was added to both the sample and the control cuvettes (ˆnal concentration, 2.5 mM), and spectra were determined at certain periods (in min) as indicated: (1) proximately 1.5 mM, and the activity decreased with increases in the concentration of the substrate up to 10 mM. At low concentrations of cysteine, the apparent Km for L-cysteine and Vmax were estimated to be 0.46 mM and 0.23 mmol W min W mg of protein, respectively, in a Lineweaver-Burk plot (data not shown).…”

“…4) The multicatalytic property of this enzyme is shared by the CTT g-lyases from other sources. [5][6][7] L-Cysteine can also be another substrate of the CTT g-synthase reaction catalyzed by the same enzyme from various sources, [4][5][6]8) together with L-homoserine (or its analogues), at high concentrations that are su‹cient to inhibit the b-and g-elimination reactions. 2,3) On the other hand, L-cysteine forms thiazolidine with pyridoxal 5?-phosphate (PLP), 9,10) and it can be used to prepare apoproteins of some enzymes having PLP as the cofactor.…”

“…13) CTT g-lyase of S. cerevisiae (a tetramer of identical subunits having a MW of 43,000 8) ) was puriˆed from transformed E. coli cells as described previously. 8) Elimination reactions with amino acids as substrates were carried out, unless otherwise stated, at 309 C for 30 min, in 1 ml of a reaction mixture containing 0.1 M potassium phosphate buŠer (pH 8.0) and 0.2 mM PLP, using an appropriate amount of the enzyme (0.011-0.033 mg of protein). When cysteine was used as the substrate, dithiothreitol (DTT) was used at 1 mM in the reaction mixture to minimize interference by cystine produced during the incubation.…”