Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/136666
Citations | ||
Scopus | Web of Science® | Altmetric |
---|---|---|
?
|
?
|
Type: | Journal article |
Title: | Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering |
Author: | Coleman, T. Lee, J. Kirk, A.M. Doherty, D.Z. Podgorski, M.N. Pinidiya, D.K. Bruning, J.B. De Voss, J.J. Krenske, E.H. Bell, S.G. |
Citation: | Chemistry: A European Journal, 2022; 28(67):1-13 |
Publisher: | Wiley |
Issue Date: | 2022 |
ISSN: | 0947-6539 1521-3765 |
Statement of Responsibility: | Tom Coleman, Joel Z. H. Lee, Alicia M. Kirk, Daniel Z. Doherty, Matthew N. Podgorski, Dilshi K. Pinidiya, John B. Bruning, James J. De Voss, Elizabeth H. Krenske, and Stephen G. Bell |
Abstract: | The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxysubstituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3’-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4- phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogenbonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2’- hydroxy-, 3’-hydroxy- and 4’-hydroxy metabolites in a 83:9:8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur. |
Keywords: | cytochromes; enzyme mechanisms; metalloenzymes; molecular dynamics; X-ray crystallography |
Description: | Published 1 December 2022 |
Rights: | © 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
DOI: | 10.1002/chem.202201895 |
Grant ID: | http://purl.org/au-research/grants/arc/DP140103229 http://purl.org/au-research/grants/arc/DP180103047 http://purl.org/au-research/grants/arc/FT140100355 |
Published version: | http://dx.doi.org/10.1002/chem.202201895 |
Appears in Collections: | Molecular and Biomedical Science publications |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
hdl_136666.pdf | Published version | 2.87 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.