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

Abstract

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) 1 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 enz...

Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site‐Saturated Libraries

“…[16] The co-crystal structures of wild-type PETN reductase and mutants W102F and W102Y complexed with picric acid were determined previously. [30] A comparison of the structures showed no major structural changes, however, the conversion of W102 to phenylalanine removed the hydrogen bond between W102 NE1 and Q60 NE2 present in the wild-type structure. [24] Solution studies indicated that picric acid was bound more tightly to the active site of the two mutant enzymes, with no significant impairment of FMN reduction by NADPH.…”

The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis

“…Expression of pentaerythritol tetranitrate reductase in tobacco (Nicotiana tabacum) confers both resistance to, and the ability to transform, TNT (French et al, 1999). OPR1, OPR2, and OPR3 share 43%, 44%, and 36% identity, respectively, with pentaerythritol tetranitrate reductase, and all possess the conserved active site amino acids crucial for TNT transformation by pentaerythritol tetranitrate reductase and other members of the Old Yellow Enzyme family (Snape et al, 1997;French et al, 1998;Khan et al, 2004), suggesting that they are capable of transforming TNT.…”

Role of Active Site Residues and Solvent in Proton Transfer and the Modulation of Flavin Reduction Potential in Bacterial Morphinone Reductase

“…The corresponding residue in PETN reductase plays a major role in directing reactivity against nitroaromatic substrates along nitro reduction and hydride transfer pathways, the latter generating a Meisenheimer⅐hydride complex of the nitroaromatic substrate. High resolution structural studies indicate that in PETN reductase the tryptophan residue adopts multiple conformational states influenced by ligand (nitroaromatic) binding (38). The Meisenheimer⅐hydride pathway for nitroaromatic degradation is restricted to PETN reductase (22,39), prompting the question as to the role of the conserved tryptophan residue in the remaining members of the OYE family.…”

“…Our work described in this report has demonstrated that the conserved tryptophan residue in the active site of OYE family members is not essential for the reduction of ␣/␤ unsaturated carbonyl compounds, although in PETN reductase it plays a major role in pathway selection for the degradation of nitroaromatic compounds presumably by influencing the geometry of attack of the hydride ion on the aromatic nucleus of these substrates (38). OYE and MR are known to reduce nitroaromatic substrates, but reduction of these compounds does not proceed through the Meisenheimer reduction pathway (22).…”