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J. Biol. Chem., Vol. 279, Issue 29, 30563-30572, July 16, 2004

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Atomic Resolution Structures and Solution Behavior of Enzyme-Substrate Complexes of Enterobacter cloacae PB2 Pentaerythritol Tetranitrate Reductase

MULTIPLE CONFORMATIONAL STATES AND IMPLICATIONS FOR THE MECHANISM OF NITROAROMATIC EXPLOSIVE DEGRADATION^*

Huma Khan, Terez Barna, Richard J. Harris, Neil C. Bruce¶, Igor Barsukov, Andrew W. Munro, Peter C. E. Moody, and Nigel S. Scrutton, A Lister Institute Research Professor||

From the Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom and the ^¶Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom

The structure of pentaerythritol tetranitrate (PETN) reductase in complex with the nitroaromatic substrate picric acid determined previously at 1.55 Å resolution indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate. The data suggested the presence of an unusual bond between substrate and the tryptophan side chain. Herein, we have extended the resolution of the PETN reductase-picric acid complex to 0.9 Å. This high-resolution analysis indicates that the active site is partially occupied with picric acid and that the anomalous density seen in the original study is attributed to the population of multiple conformational states of Trp-102 and not a formal covalent bond between the indole ring of Trp-102 and picric acid. The significance of any interaction between Trp-102 and nitroaromatic substrates was probed further in solution and crystal complexes with wild-type and mutant (W102Y and W102F) enzymes. Unlike with wild-type enzyme, in the crystalline form picric acid was bound at full occupancy in the mutant enzymes, and there was no evidence for multiple conformations of active site residues. Solution studies indicate tighter binding of picric acid in the active sites of the W102Y and W102F enzymes. Mutation of Trp-102 does not impair significantly enzyme reduction by NADPH, but the kinetics of decay of the hydride-Meisenheimer complex are accelerated in the mutant enzymes. The data reveal that decay of the hydride-Meisenheimer complex is enzyme catalyzed and that the final distribution of reaction products for the mutant enzymes is substantially different from wild-type enzyme. Implications for the mechanism of high explosive degradation by PETN reductase are discussed.

Received for publication, March 31, 2004

The atomic coordinates and structure factors (code 1vyr, 1vyp, and 1vys) 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 funded by grants from the UK Biotechnology and Biological Sciences Research Council, the Wellcome Trust, and the Lister Institute of Preventive Medicine. 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 first two authors contributed equally to the work.

^|| To whom correspondence should be addressed. Tel.: 44-116-223-1337; Fax: 44-116-252-3369; E-mail: nss4{at}le.ac.uk.

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