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|Title:||The role of active site residues in cytochrome P450 BM3 from Bacillus megaterium|
|Authors:||Oliver, Catherine Frances.|
|Presented at:||University of Leicester|
|Abstract:||Cytochrome P450 BM3 from Bacillus megaterium represents a novel form of P450 consisting of a single polypeptide chain incorporating both reductase and cytochrome P450, reminiscent of mammalian P450 systems. The crystal structure highlighted two important active site residues; arginine 47, postulated to interact with the carboxylate group on the fatty acid substrate, and phenylalanine 87 which was possibly involved in creating a lipophilic pocket where the terminal methyl of the fatty acid substrate is sequestered and protected from hydroxylation.;Arginine 47 to glutamate (R47E) mutation resulted in a change from a positive to a negative charge and a decrease in the overall length of the side chain at this position. The kcat, and km and K4 were determined using fatty acids and trimethylammonium compounds, the latter being better substrates for the R47 proteins. Complete reaction mixtures for wild type and R47E mutant were analysed by NMR to confirm the presence of hydroxylated products. Paramagnetic relaxation measurements established that laurate bound closer to the iron of the mutant protein for both the ferric and ferrous state.;The arginine 47 to cysteine (R47C) mutation investigated the effect of complete removal of any charge and allowed chemical modification with either iodoacetic acid to recreate a "glutamic acid" or iodoproprionic acid creating an "unnatural" amino acid side chain at this position. The chemically modified proteins were kinetically characterised using lauric acid and all the trimethylammonium compounds as substrates.;The last mutation was phenylalanine 87 to alanine (F87A). Products of hydroxylation were separated using reverse phase HPLC and then examined by NMR. The F87A mutation's predominant product was terminally hydroxylated lauric acid, as opposed to -1 hydroxylated product with the wild type enzyme. Paramagnetic relaxation measurements showed in the reduced protein, the substrate's terminal methyl group was bound 3.1 A from the iron making it available for hydroxylation.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, MRC Toxicology Unit|
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