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Type: Theses
Title: Utilising CYP199A4 from Rhodopseudomonas palustris HaA2 for biocatalysis and mechanistic studies
Author: Chao, Rebecca
Issue Date: 2016
School/Discipline: School of Physical Sciences
Abstract: The cytochrome P450 enzyme CYP199A4 from Rhodopseudomonas palustris strain HaA2 is highly specific for the regioselective oxidation of para-substituted benzoic acids. A selection of these compounds was tested with the enzyme with the aim of investigating the mechanism of different P450-catalysed reactions. These studies revealed that the binding affinity and oxidative activity of CYP199A4 is influenced by the substituent at the para-position, and that to the enzyme’s known oxidative activities (demethylation, hydroxylation, heteroatom oxidation and desaturation) can be added alkene epoxidation, alkyne oxidation and aldehyde oxidation. The active oxidants involved in these CYP199A4-catalysed oxidations were investigated using two active site mutants at the conserved acid-alcohol pair, T252A CYP199A4 [CYP199A4 subscript] and D251N CYP199A4 [CYP199A4 subscript], which should disrupt different steps of the catalytic cycle. There was a general increase in hydrogen peroxide uncoupling in the T252A CYP199A4 [CYP199A4 subscript] mutant but significant levels of product formation were observed with each substrate. The D251N mutation reduced the activity of the enzyme dramatically in all but one case, suggesting that this mutation interferes with proton delivery as expected. The elevated rate of 4-ethynylbenzoic acid oxidation by T252A CYP199A4 [CYP199A4 subscript] when compared to the wild-type enzyme suggested the involvement of Cpd 0 in alkyne oxidation, while a reduction in activity with 4-methoxybenzoic acid implicated Cpd I in demethylation. Additionally, the notable increase in product formation and coupling efficiency of D251N CYP199A4 [CYP199A4 subscript] with 4-formylbenzoic acid suggested the involvement of the peroxo-anion in aldehyde oxidation. Larger cinnamic acids and closely related substrates were also investigated with CYP199A4. The binding affinity and oxidative activity of the enzyme decreased in the order 4-methoxybenzoic acid > 4-methoxycinnamic acid > 3-(4- methoxyphenyl)propionic acid > 4-methoxyphenylacetic acid, highlighting its selectivity for a planar, benzoic acid- or cinnamic acid-like framework. The exclusive oxidation of cinnamic acids and related derivatives at the para-position further demonstrated the high regioselectivity of CYP199A4. While CYP199A4 exhibited low oxidation activity towards para-methoxy substituted benzene derivatives, considerably higher levels of activity reminiscent of the demethylation of 4-methoxybenzoic acid were observed for the Ser244 → Asp244 (S244D) mutant of CYP199A4. The exclusive demethylation of the para-methoxy substituted benzenes by S244D revealed that the regioselectivity of CYP199A4 oxidation is maintained in this mutant. The regioselectivity of the S244D mutant was further investigated using a selection of methyl- and ethyl-substituted derivatives. The methyl analogues were exclusively oxidised at the para-position to a single α-hydroxylation product. α-Hydroxylation and Cα [α subscript] -Cᵦ desaturation products were generated in the turnovers of the ethyl derivatives. The alcohol was formed with high stereoselectivity. The electronic properties of the ethyl substrates were found to influence the ratio of hydroxylation/desaturation product, with the more electron donating substrates giving rise to a greater proportion of the latter. This suggested the involvement of a cationic intermediate in CYP199A4- catalysed desaturation.
Advisor: Bell, Stephen G.
Abell, Andrew David
Dissertation Note: Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2016.
Keywords: P450s
cinnamic acid
benzoic acid
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
DOI: 10.4225/55/58364d44b29bd
Appears in Collections:Research Theses

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