Comparing and Combining Alternative Strategies for Enhancing Cytochrome P450 Peroxygenase Activity

被引:1
作者
Podgorski, Matthew N. [1 ]
Bell, Stephen G. [1 ]
机构
[1] Univ Adelaide, Discipline Chem, Adelaide, SA 5005, Australia
基金
澳大利亚研究理事会;
关键词
biocatalysis; cytochrome P450 enzymes; hemeenzymes; peroxygenases; protein engineering; X-ray crystallography; MACROMOLECULAR CRYSTALLOGRAPHY COMMUNITIES; HYDROGEN-PEROXIDE; P450; SUBSTRATE; CYP199A4; OXIDATION; IDENTIFICATION; SULFOXIDATION; HYDROXYLATION; ACTIVATION;
D O I
10.1021/acscatal.4c07486
中图分类号
O64 [物理化学(理论化学)、化学物理学];
学科分类号
070304 ; 081704 ;
摘要
Cytochrome P450 monooxygenase (CYP) enzymes have advantageous properties over chemical catalysts. However, it is often not feasible to use CYPs in larger-scale synthesis as they require additional cofactors (NAD(P)H) and electron transfer proteins. This could be overcome by transforming CYPs into peroxygenases that use H2O2. Recently, multiple strategies have been reported for converting CYPs into peroxygenases. Mutating the residues of the acid-alcohol pair in the oxygen-binding groove to those found in natural peroxygenases can promote the desired H2O2-driven activity. Another strategy is to enlarge the enzyme's solvent channels to allow H2O2 easier access into the active site, to enhance peroxygenase activity. Here, we evaluate these different strategies by comparing the peroxygenase activities of the double I-helix mutant D251Q/T252E (the QE mutant) and the F182A mutant of the bacterial enzyme CYP199A4. We also assess whether the peroxygenase activity can be further improved by combining these mutations (to give the F182AQE mutant). The F182A mutant exhibited the highest activity toward a selection of smaller substrates that undergo O-demethylation, S-oxidation, and epoxidation reactions. All the mutants converted 4-vinylbenzoic acid into the (S)-epoxide, with the F182A mutant having the highest stereoselectivity (>99% ee). The F182A mutant was unable to oxidize 4-t-butylbenzoic acid, while the F182AQE mutant could with high activity. The F182A mutation was found to substantially alter the selectivity of the reaction with 4-ethylbenzoic acid, increasing hydroxylation activity over desaturation. The F182A mutant catalyzed significant further oxidation reactions of the primary metabolites before all the substrate had been consumed, demonstrating a relaxed substrate specificity. X-ray crystal structures of the F182A and F182AQE mutants with the substrates revealed changes in substrate binding and solvent access providing insights into these experimental observations.
引用
收藏
页码:5191 / 5210
页数:20
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