新型争论贪噬菌Variovorax paradoxus S110的细胞色素P450酶的异源表达及催化特性分析

Expression and characterization of a novel cytochrome P450 enzyme from Variovorax paradoxus S110

摘要:

细胞色素P450单加氧酶 (Cytochrome P450 monooxygenases) 是一种广谱催化剂，可以催化多种类型反应而参与生物体外源物质代谢与天然产物的合成。为丰富P450作为合成生物学的酶元件库，并探索新型催化反应，利用生物信息学手段从争论贪噬菌Variovorax paradoxus S110中挖掘出一种新型电子自供体细胞色素P450VpMO单加氧酶，属于CYP116B家族，它可以在大肠杆菌Escherichia coli异源可溶表达。酶学性质研究表明P450VpMO最适pH和最适温度分别为8.0和45 ℃，并且在温度低于35 ℃时具有良好的稳定性，Km值为0.458 mmol/L，kcat为2.438 min–1；重要的是重组P450VpMO可以催化一系列包含污染物的含甲氧基底物进行脱甲基反应，其中对4-甲氧基苯乙酮的脱甲基反应转化率高达91%。相比于其他CYP116B家族的P450酶，P450VpMO表现出较强的酶活性，这为后期进一步研究P450VpMO提供了基础。

Abstract:

Cytochrome P450 monooxygenases as powerful biocatalysts catalyze a wide range of chemical reactions to facilitate exogenous substances metabolism and biosynthesis of natural products. In order to explore new catalytic reactions and increase the number of P450 biocatalysts used in synthetic biology, a new self-sufficient cytochrome P450 monooxygenase (P450VpMO), belongs to CYP116B class, was mined from Variovorax paradoxus S110 genome and expressed in Escherichia coli. Based on characterization of the enzymatic properties, it shows that the optimal pH and temperature for P450VpMO reaction activity are 8.0 and 45 °C, respectively. P450VpMO is relatively stable at temperatures below 35 °C. The Km and kcat of P450VpMO toward 4￠-Methoxyacetophenone are 0.458 mmol/L and 2.438 min-1, respectively. Importantly, P450VpMO was able to catalyze the demethylation reaction for a range of substrates containing methoxy group. Its demethylation reactivity is reasonably better than other P450s belongs to CYP116B class, particularly, for 4-methoxyacetophenone with a great conversion efficiency at 91%, showing that P450VpMO could be used as a great biocatalyst candidate for further analysis.