title: L-cysteine desulfidase: an [4Fe-4S] enzyme isolated from Methanocaldococcus jannaschii that catalyzes the breakdown of L-cysteine into pyruvate, ammonia, and sulfide.
abstract: A [4Fe-4S] enzyme that decomposes L-cysteine to hydrogen sulfide, ammonia, and pyruvate has been isolated and characterized from Methanocaldococcus jannaschii. The sequence of the isolated enzyme demonstrated that the protein was the product of the M. jannaschii MJ1025 gene. The protein product of this gene was recombinantly produced in Escherichia coli and purified to homogeneity. Both the isolated and recombinant enzymes are devoid of pyridoxal phosphate (PLP) and are rapidly inactivated upon exposure to air. The air-inactivated enzyme is activated by reaction with Fe2+ and dithiothreitol in the absence of air. The air-inactivated enzyme contains 3 mol of iron per subunit (43 kDa, SDS gel electrophoresis), and the native enzyme has a measured molecular mass of 135 kDa (gel filtration), indicating it is a trimer. The enzyme is very specific for L-cysteine, with no activity being detected with D-cysteine, L-homocysteine, 3-mercaptopropionic acid (cysteine without the amino group), cysteamine (cysteine without the carboxylic acid), or mercaptolactate (the hydroxyl analogue of cysteine). The activity of the enzyme was stimulated by 40% when the enzyme was assayed in the presence of methyl viologen (4 mM) and inhibited by 70% when the enzyme was assayed in the presence of EDTA (7.1 mM). Preincubation of the enzyme with iodoacetamide (17 mM) completely abolishes activity. The enzymatic activity has a half-life of 8 or 12 min when the enzyme is treated at room temperature with 0.42 mM N-ethylmaleimide (NEM) or 0.42 mM iodoacetamide, respectively. MALDI analysis of the NEM-inactivated enzyme showed Cys25 as the site of alkylation. Site-directed mutagenesis of each of four of the cysteines conserved in the orthologues of the enzyme reduced the catalytic efficiency and thermal stability of the enzyme. The enzyme was found to catalyze exchange of the C-2 hydrogen of the L-cysteine with solvent. These results are consistent with three of the conserved cysteines being involved in the formation of the [4Fe-4S] center and the thiolate of Cys25 serving as a base to abstract the alpha-hydrogen in the first step of the elimination. Although the enzyme has no sequence homology to any known enzymes, including the non-PLP-dependent serine/threonine dehydratases or aconitases, the mechanisms of action of all of these enzymes are similar, in that each catalyzes an alpha,beta-elimination reaction adjacent to a carboxylate group. It is proposed that the enzyme may be responsible for the production of sulfide required for the biosynthesis of iron-sulfur centers in this archaea. A mechanism of action of the enzyme is proposed.
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0308, USA.
authors.forename: Shih-I
authors.initials: SI
authors.lastname: Tchong
authors.forename: Huimin
authors.initials: H
authors.lastname: Xu
authors.forename: Robert H
authors.initials: RH
authors.lastname: White
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D003542:Cystathionine gamma-Lyase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D008401:Gas Chromatography-Mass Spectrometry; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013440:Sulfides / Q000378:metabolism*; D013696:Temperature
chemical_list: D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D013440:Sulfides; D000641:Ammonia; D019289:Pyruvic Acid; D013437:Carbon-Sulfur Lyases; D003542:Cystathionine gamma-Lyase; D003545:Cysteine