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J. Biol. Chem., Vol. 278, Issue 44, 43682-43690, October 31, 2003

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Crystal Structure of Escherichia coli PdxA, an Enzyme Involved in the Pyridoxal Phosphate Biosynthesis Pathway^*

J. Sivaraman, Yunge Li, Jerel Banks¶, David E. Cane, Allan Matte||, and Miroslaw Cygler**

From the Biotechnology Research Institute, NRC, and the Montreal Joint Centre for Structural Biology, Montreal, Quebec H4P 2R2, Canada and the Department of Chemistry, Brown University, Providence, Rhode Island 02912-9108

Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-L-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP⁺ or NAD⁺ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn²⁺. The protein forms tightly bound dimers. Each monomer has an //-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn²⁺ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp²⁴⁷ and Asp²⁶⁷, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn²⁺ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.

Received for publication, June 16, 2003 , and in revised form, July 24, 2003.

The atomic coordinates and structure factors (codes 1PTM, 1PS7, and 1PS6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work supported in part by Canadian Institutes of Health Research Grant 200103GSP-90094-GMX-CFAA-19924 (to M. C.) and National Institutes of Health Grant GM30301 (to D. E. C.). 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.

^¶ Recipient of Ruth L. Kirschstein National Research Service Award Predoctoral Fellowship GM20096.

|| To whom correspondence may be addressed: Biotechnology Research Institute, NRC, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-2557; Fax: 514-496-5143; E-mail: allan{at}bri.nrc.ca. ^** To whom correspondence may be addressed: Biotechnology Research Institute, NRC, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-6321; Fax: 514-496-5143; E-mail: mirek{at}bri.nrc.ca.

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