1n2t
From Proteopedia
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C-DES Mutant K223A with GLY Covalenty Linked to the PLP-cofactor
Overview
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, alpha-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-DES(K223A) 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 beta-elimination reaction, leading to the formation of a hydrophobic pocket that stabilizes the, enolimine tautomer of the aminoacrylate and the cysteine persulfide, product.
About this Structure
1N2T is a Single protein structure of sequence from Synechocystis sp. pcc 6803 with K, PLP and GLY as ligands. Full crystallographic information is available from OCA.
Reference
Snapshots of the cystine lyase C-DES during catalysis. Studies in solution and in the crystalline state., Kaiser JT, Bruno S, Clausen T, Huber R, Schiaretti F, Mozzarelli A, Kessler D, J Biol Chem. 2003 Jan 3;278(1):357-65. Epub 2002 Oct 16. PMID:12386155
Page seeded by OCA on Sat Nov 24 22:27:56 2007
