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          Institute: MPI für medizinische Forschung     Collection: Abteilung Molekulare Zellforschung     Display Documents

ID: 22651.0, MPI für medizinische Forschung / Abteilung Molekulare Zellforschung
Evidence for cystein persulfide as reaction product of L-Cyst(e)ine C-S-Lyase (C-DES) from synechocystis
Translation of Title:Evidence for cystein persulfide as reaction product of L-Cyst(e)ine C-S-Lyase (C-DES) from synechocystis
Authors:Lang, Thorsten; Kessler, Dorothea
Date of Publication (YYYY-MM-DD):1999
Title of Journal:Journal of Biological Chemistry
Journal Abbrev.:J. Biol. Chem. (JBCHA3)
Issue / Number:1
Start Page:189
End Page:195
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:The pyridoxal phosphate-dependent monomeric L-cysteine/cystine C-S-lyase (C-DES), previously isolated from Synechocystis PCC 6714 by its capacity to direct [2Fe-2S] cluster assembly of ferredoxin in vitro (Leibrecht, I., and Kessler, D. (1997) J. Biol. Chem. 272, 10442-10447), has now been cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence of C-DES was found to be nearly identical (92% identity) to the open reading frame slr2143 of Synechocystis PCC 6803 and showed a more distant relationship to the NifS family of proteins (about 27% identity). Recombinant C-DES displayed activities equal to the isolate from Synechocystis in terms of the cyst(e)ine lyase reaction and holoferredoxin formation which recommended its use for functional and mechanistic studies. Investigation of the substrate spectrum for beta -elimination found L-cysteine to be a poor substrate (kcat approx 0.15 s-1) in contrast to L-cystine (kcat = 36 s-1) and several related compounds. Of these compounds, desaminocystine (S-(carboxyethylthio)-L-cysteine) was used for C-DES-mediated persulfide generation. Stabilization of the linear persulfide 3-(disulfanyl)-propionic acid was achieved by cyclization as a novel intramolecular trapping reaction; this yielded 1,2-dithiolan-3-one which was isolated and identified by chemical analyses. Continuing interest in structure and function of iron-sulfur proteins (1) has recently been extended to the biosynthetic problem of how the Fe-S cluster moieties are introduced into the proteins. Genetic studies on the biosynthesis of the nitrogenase metalloclusters in Azotobacter vinelandii revealed nifS as essential for assembly of the [4Fe-4S] cluster of the iron protein component (2). NifS protein was subsequently identified as a L-cysteine desulfurase, yielding alanine and sulfur as products (3). A specific cysteinyl residue of NifS serves as the primary sulfur acceptor (4) and has been suggested as persulfidic sulfur donor compound for nitrogenase apo-iron protein. A more general role for the NifS-type chemistry might be indicated by the fact that genes homologous to nifS have been found in a variety of organisms (3, 5) and that a NifS-like protein has been isolated from Escherichia coli by its capability to restore the activity of damaged dihydroxy acid dehydratase (6), which is an oxidation sensitive [4Fe-4S] enzyme. Our work, designed to identify proteins involved in ferredoxin [2Fe-2S] cluster assembly with the cyanobacterium Synechocystis PCC 6714 as a model led to the straightforward purification of a L-cysteine/cystine C-S-lyase (7). This lyase, named C-DES,1 is distinguished from NifS in being monomeric, insensitive to thiol-alkylating reagents and in producing pyruvate and sulfide from cysteine (instead of alanine and sulfur). Remarkably, C-DES was isolated from Synechocystis extract by use of a holoferredoxin formation assay (7) despite the occurrence of 3 nifS-type sequences in the genome as proven for the related strain Synechocystis PCC 6803 (8). With the assay system employed, 1 mol of [2Fe-2S] ferredoxin was formed per 2 mol of cysteine utilized (Equation 1; Ref. 7). Cysteine cleavage could also be observed in the absence of apoferredoxin which made obvious that beta -elimination and cluster formation are not necessarily coupled processes. Investigation of the lyase reaction per se revealed that C-DES strongly preferred L-cystine to L-cysteine (7). beta -Elimination of cystine should yield cysteine persulfide as an unstable, substrate-derived S0 compound. For accumulation and chemical characterization of the postulated persulfide molecule substantial amounts of C-DES were required. We here report the cloning and sequencing of the C-DES gene and the overproduction of the gene product in E. coli. Using the recombinant enzyme we have investigated the specificity of C-DES with respect to the cyst(e)ine substrate. Formation of a substrate-derived persulfide was established using desaminocystine (S-(2-carboxyethylthio)-L-cysteine). The resultant 3-(disulfanyl)-propionic acid became stable by cyclization and was identified as 1,2-dithiolan-3-one. Part of this work was reported at the GBM Fall Meeting (T?bingen, Federal Republic of Germany, Ref. 9).
External Publication Status:published
Document Type:Article
Communicated by:Wulf Kaiser
Affiliations:MPI für medizinische Forschung/Ehem. Abteilung Molekulare Zellforschung/
Identifiers:URI: [Full text]
URI: [Abstract]
URI: [Fulltext PDF]
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