Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

Snape, Jason; Walkley, Neal A.; Morby, Andrew P.; Nicklin, S.; White, Graham F.

doi:10.1128/jb.179.24.7796-7802.1997

Cited by 79 publications

(67 citation statements)

References 39 publications

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“…The OYE-catalyzed NADPHdependent denitration of organic nitrates GTN and PDN is a reduction reaction producing nitrite, as demonstrated previously for OYE homologs (11)(12). This property was confirmed further in this study by detecting and measuring nitrite, using the diazo-coupling method (22).…”

Section: Discussionsupporting

confidence: 61%

“…Dichloromethane extracts of the reaction media were dried and analyzed by NMR to identify the degradation products of GTN and PDN. (25) to identify denitration products in the reaction with the GTN reductase from Agrobacterium radiobacter (12).…”

Section: Nmr Analysis Of the Reaction Productsmentioning

confidence: 99%

“…During the last few years, an increasing number of proteins have been isolated from bacterial, yeast, and plant sources, which share significant sequence identity with OYE (10)(11)(12)(13)(14)(15)(16)(17)(18). A number of these enzymes from microbial sources have been shown to catalyze the degradation of nitrate esters (19).…”

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confidence: 99%

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Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Meah

Brown

Chakraborty

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25°C of 145 M ؊1 s ؊1 and 5.8 M ؊1 s ؊1 , respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme-substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent Kd values of the enzyme-substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzymecatalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the freeflavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4 -5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

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Section: Discussionsupporting

confidence: 61%

Section: Nmr Analysis Of the Reaction Productsmentioning

confidence: 99%

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Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Meah

Brown

Chakraborty

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

mentioning

confidence: 99%

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

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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...

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“…The OYE family has grown steadily in recent years and now features the bacterial nitro-ester reductases PETN reductase (8), glycerol trinitrate reductase (9), XenA/B reductase (10), the bacterial morphinone reductase (11), the YqjM from Bacillus subtilis (41), the plant oxophytodienoic acid reductases (12,13), several yeast OYEs (14 -17), and an enzyme involved in prostaglandin synthesis in Trypanosoma cruzi (18). Although these enzymes originate from different organisms and catalyze different reactions, they share several common functional characteristics.…”

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confidence: 99%

Ligand-induced Conformational Changes in the Capping Subdomain of a Bacterial Old Yellow Enzyme Homologue and Conserved Sequence Fingerprints Provide New Insights into Substrate Binding

Hemel

Brigé

Savvides

et al. 2006

Journal of Biological Chemistry

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We have recently reported that Shewanella oneidensis, a Gram-negative ␥-proteobacterium with a rich arsenal of redox proteins, possesses four old yellow enzyme (OYE) homologues. Here, we report a series of high resolution crystal structures for one of these OYEs, Shewanella yellow enzyme 1 (SYE1), in its oxidized form at 1.4 Å resolution, which binds a molecule of PEG 400 in the active site, and in its NADH-reduced and p-hydroxybenzaldehyde-and p-hydroxyacetophenone-bound forms at 1.7 Å resolution. Although the overall structure of SYE1 reveals a monomeric enzyme based on the ␣ 8 ␤ 8 barrel scaffold observed for other OYEs, the active site exhibits a unique combination of features: a strongly butterfly-bent FMN cofactor both in the oxidized and NADH-reduced forms, a collapsed and narrow active site tunnel, and a novel combination of conserved residues involved in the binding of phenolic ligands. Furthermore, we identify a second p-hydroxybenzaldehyde-binding site in a hydrophobic cleft next to the entry of the active site tunnel in the capping subdomain, formed by a restructuring of Loop 3 to an "open" conformation. This constitutes the first evidence to date for the entire family of OYEs that Loop 3 may indeed play a dynamic role in ligand binding and thus provides insights into the elusive NADH complex and into substrate binding in general. Structure-based sequence alignments indicate that the novelties we observe in SYE1 are supported by conserved residues in a number of structurally uncharacterized OYEs from the ␤-and ␥-proteobacteria, suggesting that SYE1 represents a new subfamily of bacterial OYEs.

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Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

Cited by 79 publications

References 39 publications

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

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

Ligand-induced Conformational Changes in the Capping Subdomain of a Bacterial Old Yellow Enzyme Homologue and Conserved Sequence Fingerprints Provide New Insights into Substrate Binding

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