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The molecular enzymology of cytochromes P450 of biotechnological and medical interest

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thesis
posted on 15.12.2014, 10:32 by Ashley James Warman
This thesis reports the characterization of two members of a superfamily of enzymes, namely CYP51 from the human pathogen Mycobacterium tuberculosis and CYP116B1 from the soil bacterium Ralstonia metallidurans . CYP51 is one of twenty P450s encoded in the M. tuberculosis genome and has been demonstrated to demethylate sterol compounds. In this study, the spectroscopic and biophysical properties of CYP51 are extensively characterized, focussing on the unusual collapse of its carbon monoxy-ferrous form to a novel, protonated species, and interactions with a proposed physiological redox partner: a ferredoxin protein found adjacent to CYP51 in the M. tuberculosis genome. Additionally, the plausibility of this enzyme as a target for novel anti-tubercular agents, particularly azole-antifungal derivatives, is assessed. The study of CYP116B1 describes initial characterization of a novel fusion of a cytochrome P450 to its flavin mononucleotide- and iron sulphur cluster-containing redox partner protein, which is of potential biotechnological interest due to its proposed role in thiocarbamate herbicide degradation. Assessment of the spectroscopic properties of this enzyme are reported, including electron paramagnetic resonance and resonance Raman investigations of the heme macrocycle, in addition to kinetic studies of the electron transfer capabilities of the reductase component. Furthermore, the search for a substrate for this P450 and subsequent LCMS product analysis assays with the thiocarbamates EPTC and vernolate are detailed. The dissection of CYP116B1 into its constituent domains, in order to facilitate studies of the P450 and reductase components in isolation and further resolve structural, spectroscopic, and thermodynamic properties of this enzyme is also reported.

History

Date of award

01/01/2007

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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