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|Title:||Mechanistic studies of the explosive degrading enzyme, pentaerythritol tetranitrate (PETN) reductase|
|Presented at:||University of Leicester|
|Abstract:||PETN reductase is a member of the old yellow enzyme (OYE) family of flavoproteins. The enzyme is able to utilise diverse substrates such as a/b-unsaturated compounds, nitrate esters and nitroaromatic explosives, which makes it of potential interest of phytoremediation purposes. This thesis describes the characterisation of PETN reductase and investigates the role of four active site residues. Trp 102 is shown to influence the binding and transformation of TNT/picric acid. PETN reductase transforms TNT via two competing pathways; (i) nitro group reduction and (ii) direct ring reduction leading to hydride-Meisenheimer complex formation. Mutation of Trp 102 significantly affects the stability of the hydride-Meisenheimer complex, which is sufficient to affect the concentration distribution and/or nature of final TNT degradation products. The ability to produce the TNT hydride-Meisenheimer complex is abolished in the H181A and H184A mutant enzymes indicating that His 181 and His 184 determine the initial route of TNT transformation. These residues are also crucial in ligand binding. Hydride transfer to 2-cyclohexenone and nitrocyclohexene is compromised in the H181A and H184A PETN reductases, owing to the altered mode of substrate binding. Tyr compounds, however, studies presented herein reveal that his is not the case. Trp 102 and Tyr 186 are also shown to play a key role in GTN reduction. Studies on the reductive half-reaction reveal that the rate of NADPH-enzyme complex formation occurs too fast to be measured with Y186F enzyme. Conversely, no complex formation between NADPH and the H181A and H184A mutant enzymes is observed, attributed to the perturbation in NADPH binding, which favours hydride transfer. Collectively, the research described proposes interesting implications into the underlying mechanism of TNT and 2-cyclohexneone reduction by the OYE family of enzymes.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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