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Snapshots of the cystine lyase C-DES during catalysis

Kaiser, J. T., Bruno, S., Clausen, T., Huber, Robert, Schiaretti, F., Mozzarelli, A. and Kessler, D. 2002. Snapshots of the cystine lyase C-DES during catalysis. Journal of Biological Chemistry 278 (1) , pp. 357-365. 10.1074/jbc.M209862200

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Abstract

The cystine lyase (C-DES) of Synechocystis is a pyridoxal-5′-phosphate-dependent enzyme distantly related to the family of NifS-like proteins. The crystal structure of an N-terminal modified variant has recently been determined. Herein, the reactivity of this enzyme variant was investigated spectroscopically in solution and in the crystalline state to follow the course of the reaction and to determine the catalytic mechanism on a molecular level. Using the stopped-flow technique, the reaction with the preferred substrate cystine was found to follow biphasic kinetics leading to the formation of absorbing species at 338 and 470 nm, attributed to the external aldimine and the α-aminoacrylate; the reaction with cysteine also exhibited biphasic behavior but only the external aldimine accumulated. The same reaction intermediates were formed in crystals as seen by polarized absorption microspectrophotometry, thus indicating that C-DES is catalytically competent in the crystalline state. The three-dimensional structure of the catalytically inactive mutant C-DESK223A in the presence of cystine showed the formation of an external aldimine species, in which two alternate conformations of the substrate were observed. The combined results allow a catalytic mechanism to be proposed involving interactions between cystine and the active site residues Arg-360, Arg-369, and Trp-251*; these residues reorient during the β-elimination reaction, leading to the formation of a hydrophobic pocket that stabilizes the enolimine tautomer of the aminoacrylate and the cysteine persulfide product.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: American Society for Biochemistry and Molecular Biology
ISSN: 0021-9258
Last Modified: 24 Jun 2017 11:00
URI: http://orca-mwe.cf.ac.uk/id/eprint/70198

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