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Type: Thesis
Title: Harnessing P450 enzymes as biocatalysts for selective C-H bond hydroxylation
Author: Lee, Joel Hoong Zhang
Issue Date: 2018
School/Discipline: School of Physical Sciences : Chemistry
Abstract: The cytochrome P450 enzyme, CYP101B1 from Novosphingobhium aromaticivorans can catalyse the highly efficient and regioselective oxidation of norisoprenoids. However, it has lower affinity towards hydrophobic substrates. Site-saturation mutagenesis of its Histidine85 (H85) residue was carried out as the equivalent tyrosine residue in the sequence of the closely related P450cam enzyme (CYP101A1 from a Pseudomonas species) provides hydrophilic interactions with camphor. Mutagenesis of the H85 residue to a phenylalanine residue (H85F) has been reported to increase the enzyme's affnity towards small hydrophobic molecules. Eleven mutants that differ at position 85 were successfully produced and these were incorporated into two separate plasmid vectors for enzyme purification and whole-cell studies. Whole-cell oxidation of the CYP101B1 mutants with various substrates that include norisoprenoids, terpenoids, and hydrophobic molecules of varying sizes were carried out to screen if the mutants have activity towards these substrates. A number of the mutants such as H85V, H85S, H85G and H85I displayed increased product formation and altered product selectivity but most were not as effective as the WT or the H85F mutant in oxidising hydrophobic molecules. Purification of both the H85A and H85G mutants was also carried out and ferrous- CO assays showed they were functional P450 enzymes. In vitro studies with these two mutants demonstrated they have poorer binding affnity and oxidation activity towards phenylcyclohexane when compared to the WT enzyme and the H85F variant. A mutant library of rationally designed P450cam variants which have been created to increase the affinity and oxidation activity towards the monoterpene, α-pinene, was tested with other monoterpenoids and related compounds. The mutations in this library were incorporated at residues in the active site. The library was screened using a whole-cell system with five substrates of similar size and chemical functionality to camphor and pinene. These were fenchone, fenchyl acetate, isophorone, 1,8-cineole and 1,4-cineole. The screening of these mutants displayed higher selectivity for the oxidations of these substrates compared to the WT. The metabolites were produced in larger quantities for isolation and identification. Fenchone and fenchyl acetate oxidation by different mutants generated a mixture of the C5, C6 and C7 hydroxylation products with the exo face being preferred. Isophorone oxidation by the mutants F87W-Y96FL244A- V247L (WFAL) and F87W-Y96F-L244A (WFA) gave selective formation of (4R)-hydroxyisophorone. This product is valuable as a avouring agent. Several of the mutants also selectively oxidised 1,8- and 1,4-cineole at different C-H bonds. A desaturation product was observed at the isopropyl group during 1,4-cineole oxidation. Concurrent studies of isophorone and cineole oxidation by mutants of another enzyme (P450BM3) also gave selective transformation of both substrates with high yields in whole-cell turnover systems. The whole-cell oxidation of isophorone and cineole by these P450cam and P450BM3 mutants were compared to determine if oxidation activity of the two enzymes were similar. For the isophorone turnovers, the P450cam mutants resulted in greater product yields over the P450BM3 mutants, whereby most if not all substrate added was converted to product. In contrast, cineole oxidation by the P450BM3 mutants gave higher yields over the P450cam mutants. As well as oxidising norisoprenoids, CYP101B1 displayed efficient and selective oxidation of monoterpenoid acetates. P450BM3 mutants have also been developed to oxidise terpenes and norisoprenoids. A selection of avour and fragrance compounds with structures similar to norisoprenoids and monoterpenoid acetates were screened both in vitro and in vivo with CYP101B1 and P450BM3-A74G/F87V/L188Q (GVQ). CYP101B1 displayed selective oxidation towards norisoprenoids such as δ- and α- damascone. P450BM3-GVQ also showed selective oxidation for acetate compounds such as cuminyl acetate and verdyl acetate. Larger scale turnovers with these two P450 enzymes were carried out and the metabolites were identified. CYP101B1 oxidises norisoprenoids at the ring C3/C4 position and this was preserved in the oxidation of δ- and α-damascone. α-Damascone oxidation by CYP101B1 formed both C3 hydroxy diastereomers and a further oxidation product at the same position. δ-Damascone oxidation occurred across its ring C3/C4 alkene to form the epoxide. P450BM3-GVQ oxidised both cuminyl and verdyl acetate at regions distant from the acetate group. Cuminyl acetate oxidation by this mutant also generated a desaturation product at the isopropyl group.
Advisor: Bell, Stephen
Abell, Andrew
Dissertation Note: Thesis (MPhil) -- University of Adelaide, School of Physical Sciences, 2018
Keywords: P450
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