Crystal structure of pentaerythritol tetranitrate reductase: “flipped” binding geometries for steroid substrates in different redox states of the enzyme

Barna, T.; Khan, Huma; Bruce, Neil C.; Barsukov, Igor; Scrutton, Nigel S.; Moody, P.C.E.

doi:10.1006/jmbi.2001.4779

Cited by 96 publications

(165 citation statements)

References 41 publications

Supporting

Mentioning

158

Contrasting

Order By: Relevance

“…ratio of the product was 1.9, consistent with pure preparations of A-side NADP#H obtained by other workers [33]. NADP#H synthesized by this method gives rise to the expected published primary kinetic isotope effect (KIE l 7.0 ; [34]) in stopped-flow reactions with pentaerythritol tetranitrate reductase and yields a monophasic transient for flavin reduction. NADPH synthesized in the same way does not produce inhibitory products in this same reaction and likewise yields a monophasic kinetic transient.…”

Section: Synthesis Of A-side Nadp 2 Hsupporting

confidence: 88%

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Knight

Scrutton

2002

Biochemical Journal

138

View full text Add to dashboard Cite

The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP + . The second and third steps represent further reduction of the enzyme flavins and NADP + release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of

show abstract

Section: Synthesis Of A-side Nadp 2 Hsupporting

confidence: 88%

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Knight

Scrutton

2002

Biochemical Journal

138

View full text Add to dashboard Cite

show abstract

“…2 Ligand binding studies were also as performed previously with wild-type PETN reductase (14), relying on the perturbation of the flavin electronic absorption spectrum on binding ligand in the active site of PETN reductase. Data were collected in the UV-visible region (250 -600 nm), and the absorption at 518 nm plotted as a function of ligand concentration.…”

Section: Methodsmentioning

confidence: 99%

“…Crystallography-Crystals of wild-type, W102F, and W102Y PETN 2 In Ref. 14 the midpoint reduction potential for the concerted two electron reduction of wild-type PETN reductase is reported as Ϫ195 Ϯ 5 mV.…”

Section: Methodsmentioning

confidence: 99%

“…14; similar spectral changes were also observed for the W102Y and W102F enzymes. reductase in complex with picric acid were grown in the manner described previously for wild-type PETN reductase (2,14). The crystals were isomorphous with those previously described, having space group P2 1 2 1 2 1 and one molecule per asymmetric unit.…”

Section: Methodsmentioning

confidence: 99%

“…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 unusual bond between substrate and the tryptophan side chain (14).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Khan

Barna

Harris

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Enzymes, Enolate Reductases “Old Yellow Enzyme”

Hall

Yanto

Bommarius

2010

Encyclopedia of Industrial Biotechnology

View full text Add to dashboard Cite

Reported enoate reductase activity is mostly displayed by enzymes belonging to the “Old Yellow Enzyme” family of flavoproteins. They have received increased interest over the last couple of years due to their huge potential in biocatalysis. These FMN‐containing enzymes catalyze the asymmetric reduction of activated C═C bonds at the expense of a nicotinamide cofactor, producing up to two chiral centers. They are also involved in other reductive processes, such as cleavage of nitrate esters, reduction of aromatic nitro groups, and even reduction of alkynes. Increasing knowledge about this family of biocatalysts together with recent advances in biotechnology render the enoate reductases a tool of choice for industrial applications, as catalyzed reactions are usually chemo‐, regio‐ and stereo‐selective, and the enzymes most importantly display a wide substrate spectrum. With enzymatic reactions and mechanisms being generally clearly understood, the efficient production of a number of enantiopure compounds via recycling of the cofactor is now feasible.

show abstract

Crystal structure of pentaerythritol tetranitrate reductase: “flipped” binding geometries for steroid substrates in different redox states of the enzyme

Cited by 96 publications

References 41 publications

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

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

Enzymes, Enolate Reductases “Old Yellow Enzyme”

Contact Info

Product

Resources

About