The cytochrome P450 monooxygenase discovered in Labrenzia aggregata (P450_LaMO) is a self‐sufficient redox system with versatile oxygenation functions. However, its catalytic performance is severely hindered by a low reaction rate, poor electron coupling efficiency (CE) and fragile thermostability. Herein, a simple transdomain combination mutation strategy was proposed for engineering this multi‐domain P450 enzyme with redox partners fused to the heme domain. After focused mutagenesis on the heme domain, a triple mutant H3 (N119C/V264A/V437G) was hit, that improved the turnover frequency (TOF) and CE of P450_LaMO by about 7.8‐fold and 3.0‐fold, respectively. A redox domain‐based mutant with higher cytochrome c reduction activity, MR1 (M612L/K774Y), mediated more efficient electron transfer, elevated the TOF by 4.9‐fold, and the coupling efficiency by 4.2‐fold. The beneficial effect was further enhanced by combining the mutation sites from different domains, resulting in a combinatorial mutant (N119C/V264A/V437G/M612L/N694D) with a 9.1‐fold increase in coupling efficiency, 10‐fold in TOF, as well as +3.8 °C in thermostability (T₅₀¹⁰). Meanwhile, for series of tetrahydronaphthalene derivatives, this combinator showed higher hydroxylation activity. This work suggested that employing this combinatorial strategy targeting on both the redox and heme domains is efficient to improve holoenzyme activity, CE and stability of a CYP116B subfamily member from the low starting point.

中文翻译：

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通过跨域组合诱变提高CYP116B单加氧酶的催化性能

集合体Labrenzia aaggregata（P450 _{La MO}）中发现的细胞色素P450单加氧酶是具有多种充氧功能的自给自足的氧化还原系统。但是，其反应速度低，电子耦合效率（CE）差和热稳定性差，严重阻碍了其催化性能。在此，提出了一种简单的跨域组合突变策略，用于将这种多域P450酶与与血红素域融合的氧化还原伴侣进行工程改造。在针对血红素结构域进行集中诱变后，命中了一个三重突变体H3（N119C / V264A / V437G），这使P450 _{La MO}的周转频率（TOF）和CE分别提高了约7.8倍和3.0倍。具有较高细胞色素c的基于氧化还原域的突变体还原活性MR1（M612L / K774Y）介导的电子转移效率更高，TOF升高了4.9倍，耦合效率提高了4.2倍。通过组合来自不同域的突变位点，进一步增强了有益效果，从而产生了组合突变体（N119C / V264A / V437G / M612L / N694D），其偶联效率提高了9.1倍，TOF为10倍，并且热稳定性+3.8°C（T ₅₀ ¹⁰）。同时，对于一系列四氢萘衍生物，该组合物显示出更高的羟基化活性。这项工作表明，针对氧化还原和血红素结构域采用这种组合策略可有效地从低起点提高全酶活性，CE和CYP116B亚家族成员的稳定性。