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

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. Pentaerythritol tetranitrate (PETN) reductase is a member of the old yellow enzyme (OYE) family of flavoproteins and was purified from a strain of Enterobacter cloacae (strain PB2) originally isolated on the basis of its ability to utilize nitrate ester explosives such as PETN and glycerol trinitrate (GTN) as sole nitrogen source (1). The structure of PETN reductase (2) is similar to that of OYE (3) and morphinone reductase (4), confirming the close evolutionary relationship with OYE and other FMN-dependent flavoprotein oxidoreductases inferred from sequence analysis of the genes encoding these enzymes (5, 6). Consistent with this close relationship is the ability of the OYE family of enzymes to reduce a variety of cyclic enones, including 2-cyclohexenone and steroids. Some steroids act as substrates, whereas others are potent inhibitors of these enzymes. PETN reductase, and the related orthologues from strains of Pseudomonas (7) and Agrobacterium (8), show reactivity against explosive substrates. PETN reductase degrades major classes of explosive, including nitroaromatic compounds (e.g. trinitrotoluene TNT) and nitrate esters (GTN and PETN) (9–11). Degradation of TNT involves reductive hydride addition to the aromatic nucleus (Fig. 1). In the case of members of the old yellow enzyme family of enzymes that are closely related to PETN reductase, the products of TNT reduction have been shown to result from both reductive hydride addition at the aromatic nucleus and also nitro group reduction in two competing pathways in the oxidative half-reaction of the enzyme (9, 12, 13). The reaction of PETN reductase comprises two half-reactions: in the reductive half-reaction, enzyme is reduced by NADPH to yield the dihydroquinone form of the enzyme-bound FMN, and in the oxidative half-reaction the flavin is oxidized by the nitro-containing explosive substrates or cyclic enone substrates. A detailed kinetic mechanism based on stopped-flow data has been proposed (14), and recently hydride transfer in the reductive half-reaction was shown to proceed by quantum mechanical tunneling (15). The structures of PETN reductase in complex with steroid substrates and inhibitors have been determined (2), as have complexes of the enzyme with 2-cyclohexenone, the inhibitor 2,4-dinitrophenol (2,4-DNP), and the substrates TNT and picric acid (14). The 1.55 Å structure of the enzyme in complex with picric acid indicated additional electron density between the indole ring of residue Trp-102 and the nitro group at C-6 of picrate, which at this resolution suggested the presence of an * 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 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/). § The first two authors contributed equally to the work. A Lister Institute Research Professor. To whom correspondence should be addressed. Tel.: 44-116-223-1337; Fax: 44-116-252-3369; E-mail: [email protected]. 1 The abbreviations used are: PETN, pentaerythritol tetranitrate; TNT, trinitrotoluene; GTN, glycerol trinitrate; OYE, old yellow enzyme; 2,4-DNP, 2,4-dinitrophenol. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 29, Issue of July 16, pp. 30563–30572, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.