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J. Biol. Chem., Vol. 280, Issue 16, 15800-15808, April 22, 2005

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Crystal Structure of N-Succinylarginine Dihydrolase AstB, Bound to Substrate and Product, an Enzyme from the Arginine Catabolic Pathway of Escherichia coli^*

Ante Tocilj, Joseph D. Schrag, Yunge Li, Barbara L. Schneider¶, Larry Reitzer¶, Allan Matte, and Miroslaw Cygler||

From the Biotechnology Research Institute and the Montreal Joint Centre for Structural Biology, Montréal, Québec H4P 2R2, Canada and the ^¶Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson, Texas 75083-0688

The ammonia-producing arginine succinyltransferase pathway is the major pathway in Escherichia coli and related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure of N-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric / propeller fold of circularly arranged modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys–His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound the N-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.

Received for publication, December 8, 2004 , and in revised form, January 25, 2005.

^* This work was supported in part by the Office of Biological and Environmental Research and of Basic Energy Sciences of the United States Department of Energy and the National Center for Research Resources of the National Institutes of Health. This work was also supported by Canadian Institutes for Health Research Grant 200103GSP-90094-GMX-CFAA-19924 (to M. C.) and National Science Foundation Grant MCB-0323931 (to L. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 1YNF, 1YNH, and 1YNI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^|| To whom correspondence should be addressed: Biotechnology Research Institute, NRC, 6100 Royalmount Ave., Montreal, PQ H4P 2R2, Canada. Tel.: 514-496-6321; Fax: 514-496-5143; E-mail: mirek{at}bri.nrc.ca.

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