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J. Biol. Chem., Vol. 279, Issue 13, 13148-13155, March 26, 2004

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A Novel NAD-binding Protein Revealed by the Crystal Structure of 2,3-Diketo-L-gulonate Reductase (YiaK)^*

Farhad Forouhar, Insun Lee, Jordi Benach, Kaushal Kulkarni, Rong Xiao, Thomas B. Acton, Gaetano T. Montelione, and Liang Tong¶

From the Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York 10027 and the Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University and the Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, New Jersey 08854

Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-Å resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His⁴⁴ is likely the catalytic residue of the enzyme.

Received for publication, December 11, 2003 , and in revised form, January 7, 2004.

The atomic coordinates and structure factors (codes 1NXU and 1S20) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by Grant P50 GM62413 from the Protein Structure Initiative of the National Institutes of Health. 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.

^¶ To whom correspondence should be addressed. Tel.: 212-854-5203; Fax: 212-854-5207; E-mail: tong{at}como.bio.columbia.edu.

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