Evidence for cysteine persulfide as reaction product of L-Cyst(e)ine C-S-lyase (C-DES) from Synechocystis. Analyses using cystine analogues and recombinant C-DES.
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 approximately 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.
Lang
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Biochemiezentrum Heidelberg, Universität Heidelberg, D-69120 Heidelberg, Federal Republic of Germany.
Kessler
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D000595:Amino Acid Sequence; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D003553:Cystine / Q000031:analogs & derivatives*; D004269:DNA, Bacterial; D004220:Disulfides / Q000737:chemistry*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D004220:Disulfides; D011994:Recombinant Proteins; C116840:cysteine persulfide; D003553:Cystine; D013437:Carbon-Sulfur Lyases; C105473:L-cysteine-cystine C-S lyase; D003545:Cysteine