1N31

Structure of A Catalytically Inactive Mutant (K223A) of C-DES with a Substrate (Cystine) Linked to the Co-Factor

Summary for 1N31

• Entry DOI: 10.2210/pdb1n31/pdb
• Related: 1ELQ 1ELU 1N2T
• Descriptor: L-cysteine/cystine lyase C-DES, POTASSIUM ION, PYRIDOXAL-5'-PHOSPHATE, ... (5 entities in total)
• Functional Keywords: inactive mutant, substrate complex, fe-s cluster synthesis, nifs-like, lyase
• Biological source: Synechocystis sp. PCC 6714
• Total number of polymer chains: 2
• Total formula weight: 85824.86

Authors

Kaiser, J.T.,Bruno, S.,Clausen, T.,Huber, R.,Schiaretti, F.,Mozzarelli, A.,Kessler, D. (deposition date: 2002-10-25, release date: 2003-01-21, Last modification date: 2023-12-13)

Primary citation

Kaiser, J.T.,Bruno, S.,Clausen, T.,Huber, R.,Schiaretti, F.,Mozzarelli, A.,Kessler, D.
Snapshots of the Cystine Lyase "C-DES" during Catalysis: Studies in Solution and in the Crystalline State
J.Biol.Chem., 278:357-365, 2003

PubMed 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 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.

PubMed: 12386155
DOI: 10.1074/jbc.M209862200

Experimental method

X-RAY DIFFRACTION (2.2 Å)