Comparing and Combining Alternative Strategies for Enhancing Cytochrome P450 Peroxygenase Activity

• 全文链接

提高细胞色素P450过加氧酶活性的几种策略的比较与组合

关键词：

来源：

ACS Catalysis

全文链接：

//agri.nais.net.cn/topic/downloadFile/f53f9d48-d06e-420b-9373-1d2975ef6a46

来源地址：

https://pubs.acs.org/doi/10.1021/acscatal.4c07486

资源所属：

饲料用酶工程

类型：

学术文献

语种：

英语

原文发布日期：

2025-03-12

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.