Pentaerythritol tetranitrate reductase: kinetic and structural basis of reactivity with NADPH, 2-cyclohexenone, nitroesters and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidising substrates has been studied by stopped-flow spectrophotometry, redox potentiometry and X-ray crystallography. We show in the reductive half-reaction of PETN reductase that NADPH binds to form an enzyme -NADPH charge -transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the 2 electron-reduced enzyme reacts with several substrates including nitroester explosives [glycerol trinitrate (GTN) and pentaerythritol tetranitrate (PETN)], nitroaromatic explosives [trinitrotoluene (TNT) and picric acid] and α,β unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride -Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 at the electron-deficient aromatic nucleus of the substrate. Crystal structures of complexes of the oxidised enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4 dinitrophenol (2,4 DNP) is similar to that observed with picric acid and TNT. In this position the aromatic nucleus, however, is not activated for hydride transfer from the flavin N5, thus accounting for the lack of reactivity with 2,4 DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins, but at the same time highlights important differences compared with the reactivity of OYE. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid, and for the inhibition of enzyme activity with 2,4 DNP. by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Khan, Harris, Barna, Craig, Bruce, Munro, Moody & Scrutton 4 INTRODUCTION A large number of sites worldwide are contaminated with high explosives as a result of large-scale manufacturing and handling of these compounds. Bioremediation is an attractive means of decontaminating such sites (1), which has led to a search for enzymes capable of degrading high explosive compounds. We previously isolated a strain of Enterobacter cloacae (strain PB2) on the basis of its ability to utilise nitrate ester explosives such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate (GTN) as sole nitrogen source (2). The ability of Enterobacter cloacae PB2 to utilise nitrate esters as a nitrogen source is conferred by the NADPH-dependent flavoenzyme PETN reductase (3). Sequence analysis of the cloned gene encoding PETN reductase has established a close evolutionary relationship with the flavoenzyme Old Yellow Enzyme (OYE) (4), and related enzymes such as bacterial morphinone reductase (MR) (5) and the estrogen-binding protein (EBP) of Candida albicans (6). These enzymes bind a variety of cyclic enones, including 2-cyclohexenone and steroids. Some steroids act as substrates whilst others are inhibitors of both PETN reductase and OYE. We have demonstrated that PETN reductase degrades all major classes of explosive including nitroaromatic compounds [e.g. trinitrotoluene (TNT); (7-9)] and cyclic triazine explosives [e.g. Royal Demolition Explosive (RDX)], making the enzyme attractive in phytoremediation of explosive contaminated land (10). Homologues of PETN reductase from strains of Pseudomonas (11) and Agrobacterium (12) have been isolated and these enzymes also show reactivity against explosive substrates. In the case of xenobiotic reductase from Pseudomonas fluorescens I-C, the products of TNT reduction have been identified and by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Khan, Harris, Barna, Craig, Bruce, Munro, Moody & Scrutton 5 shown to proceed either by hydride addition to the aromatic nucleus or by nitro group reduction (13). The crystal structure of PETN reductase has been solved in both its oxidised and 2 electron-reduced forms (14). The structures of a number of complexed forms with both steroid substrates and inhibitors are also known (14). The enzyme is a conventional eightfold β/α barrel protein that contains a single FMN redox centre, and overall resembles the structure of OYE (15). However, the mode of steroid binding to oxidized enzyme differs from that seen with OYE in that the reactive olefinic bond in the steroid is not positioned over the flavin N5 (14). Reactions performed with ‘A-side’ deuterated nicotinamide cofactor have shown that in 2 electron-reduced PETN reductase the steroid is ‘flipped’ compared with the mode of binding to oxidized enzyme (14). In this ‘flipped’ binding mode the reactive olefinic bond is aligned with the flavin N5 atom in a geometry that is compatible with hydride transfer to the steroid substrate. Deuterium labelling methods have enabled us to assign the reactive olefinic bond as the C1-C2 bond in 1,4−androstadiene-3,17-dione and prednisone, to elucidate the stereochemistry of bond reduction and to propose a mechanism for the reduction of cyclic enones (14). Our work on the stereochemistry of olefinic bond reduction by PETN reductase again establishes a close relationship with OYE. Vaz et al (16) have shown that reduction of α,β-unsaturated carbonyl compounds by OYE proceeds by hydride transfer from the flavin N5 to the β carbon followed by proton uptake at the α carbon — a finding that is consistent with our more recent determination of the stereochemistry of bond reduction catalysed by PETN reductase. by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Khan, Harris, Barna, Craig, Bruce, Munro, Moody & Scrutton 6 In this paper we report a detailed kinetic analysis of the reaction of PETN reductase with NADPH and the substrate 2-cyclohexenone, which is used widely as a ‘generic’ substrate of the OYE family of enzymes. We also report studies of enzyme oxidation by nitroester substrates (GTN and PETN) and the nitroaromatic explosives TNT and picric acid. The structures of PETN reductase complexed with picric acid, TNT, 2-cyclohexenone and the inhibitor 2,4 dinitrophenol (2,4-DNP) are also presented, which provide atomic insight into the mechanism of nitroaromatic reduction and the reduction of α,β unsaturated carbonyl compounds. by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Khan, Harris, Barna, Craig, Bruce, Munro, Moody & Scrutton 7 EXPERIMENTAL PROCEDURES Chemicals and enzymes — Complex bacteriological media were from Unipath and all media were prepared as described by Sambrook et al (17). Mimetic Orange 2 affinity chromatography resin was from Affinity Chromatography Ltd. Q-sepharose resin was from Pharmacia. PETN reductase was prepared from E. coli JM109/pONR1 and purified as described (3) , but also incorporating a final chromatographic step using Q-sepharose (14). NADPH, glucose 6-phosphate dehydrogenase, glucose 6-phosphate, benzyl viologen, methyl viologen, 2-hydroxy-1,4-naphthaquinone, phenazine methosulfate and 2,4 DNP were from Sigma. 2-cyclohexenone was from Acros Organics. Dr S Nicklin (UK Defence and Evaluation Research Agency) supplied TNT, GTN, PETN and picric acid. The following extinction coefficients were used to calculate the concentration of substrates and enzyme: NADPH (ε340 = 6.22 x 10 M cm); PETN reductase (ε464 = 11.3 x 10 M cm); 2-cyclohexenone (ε232 = 11.0 x 10 M cm). Stock solutions of TNT (600 mM) were made up in acetone. Dilutions were then made into potassium phosphate buffer, pH 7.0, and the acetone concentration was maintained at 1 % (v/v). The presence of acetone in buffers at 1 % (v/v) was shown not to affect enzyme activity. Redox potentiometry — Redox titrations were performed within a Belle Technology glove box under a nitrogen atmosphere (oxygen maintained at <5 ppm) in 50 mM potassium phosphate buffer, pH 7.0. Anaerobic titration buffer was prepared by flushing freshly prepared buffer with oxygen-free nitrogen. PETN reductase admitted to the glove box was de-oxygenated by passing through a Biorad 10DG column, with final dilution of the eluted protein to give a concentration of ~60 μM. Solutions of benzyl viologen, by gest on N ovem er 7, 2017 hp://w w w .jb.org/ D ow nladed from Khan, Harris, Barna, Craig, Bruce, Munro, Moody & Scrutton 8 methyl viologen, 2-hydroxy-1,4-naphthaquinone and phenazine methosulfate were added to a final concentration of 0.5 μM as redox mediators for the titrations. Absorption spectra (300 750 nm) were recorded on a Varian (Cary 50 probe) UV-visible spectrophotometer, and the electrochemical potential was monitored using a Hanna instruments pH/voltmeter coupled to a Russell Pt/calomel electrode. The electrode was calibrated using the Fe(II)/Fe(III)-EDTA couple (+108 mV) as a standard. The enzyme solution was titrated electrochemically using sodium dithionite as reductant and potassium ferricyanide as oxidant, as described by Dutton (18). After the addition of each aliquot of reductant, and allowing equilibration to occur (stabilization of the observed potential), the spectrum was recorded and the potential was noted. The process was repeated at several (typically ~ 40) different potentials. In this way, a set of spectra representing reductive and oxidative titrations was obtained. Small corrections were made for any drift in the baseline by correcting the absorbance at 750 nm to zero. The observed potentials were corrected to those for the standard hydrogen electrode (SHE) (Pt/calomel + 244 mV). Data manipulation and analysis were performed using Origin software (Microcal). Absorbance values at wavelengths of 468 nm (close to the oxidized flavin maximim) were plotted against potential. Data were fitted using Eq. 1, which represents a concerted 2-electron redox process derived by extension to the Nernst equation and the Beer-Lambert Law, as described previously (18): ( ) ( ) 5 . 29 / 5 . 29 / 468 12 12 10 1 ) 10 ( E E E E b a A − −