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J. Biol. Chem., Vol. 282, Issue 11, 8487-8497, March 16, 2007

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Crystal Structure of Escherichia coli Ketopantoate Reductase in a Ternary Complex with NADP⁺ and Pantoate Bound

SUBSTRATE RECOGNITION, CONFORMATIONAL CHANGE, AND COOPERATIVITY^*

Alessio Ciulli, Dimitri Y. Chirgadze, Alison G. Smith^¶, Tom L. Blundell, and Chris Abell¹

From the University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, United Kingdom, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom, and ^¶Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom

Ketopantoate reductase (KPR, EC 1.1.1.169 [EC] ) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate, an essential step for the biosynthesis of pantothenate (vitamin B₅). Inhibitors of the enzymes of this pathway have been proposed as potential antibiotics or herbicides. Here we present the crystal structure of Escherichia coli KPR in a precatalytic ternary complex with NADP⁺ and pantoate bound, solved to 2.3Å of resolution. The asymmetric unit contains two protein molecules, each in a ternary complex; however, one is in a more closed conformation than the other. A hinge bending between the N- and C-terminal domains is observed, which triggers the switch of the essential Lys¹⁷⁶ to form a key hydrogen bond with the C2 hydroxyl of pantoate. Pantoate forms additional interactions with conserved residues Ser²⁴⁴, Asn⁹⁸, and Asn¹⁸⁰ and with two conservatively varied residues, Asn¹⁹⁴ and Asn²⁴¹. The steady-state kinetics of active site mutants R31A, K72A, N98A, K176A, S244A, and E256A implicate Asn⁹⁸ as well as Lys¹⁷⁶ and Glu²⁵⁶ in the catalytic mechanism. Isothermal titration calorimetry studies with these mutants further demonstrate the importance of Ser²⁴⁴ for substrate binding and of Arg³¹ and Lys⁷² for cofactor binding. Further calorimetric studies show that KPR discriminates binding of ketopantoate against pantoate only with NADPH bound. This work provides insights into the roles of active site residues and conformational changes in substrate recognition and catalysis, leading to the proposal of a detailed molecular mechanism for KPR activity.

Received for publication, December 5, 2006 , and in revised form, January 4, 2007.

The atomic coordinates and structure factors (code 2OFP) 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 the Biotechnology and Biological Sciences Research Council. 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1, Tables S1 and S2, and Movies S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 44-1223-336405; Fax: 44-1223-336362; E-mail: ca26{at}cam.ac.uk.

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