Enhancing the Catalytic Performance of a CYP116B Monooxygenase by Transdomain Combination Mutagenesis

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 degrees C in thermostability (T-50(10)). 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.