Data Mining And Catalytic Performance Of A New Self-sufficient Monooxygenase In CYP116B Family

Herein, we presented the first example of combining the power of discovering naturally evolved P450s through genome mining and the evolutionary advantages of P450-PDOR fusion enzyme, with the purpose of access monooxgenase with novel substrate specificities. Successfully, a novel self-sufficient monooxygenase (P450LaMo) was discovered from Labrenzia aggregate for the first time by genomic data mining. It was identified as a member of CYP116B subfamily, with60%identity to P450RhF from Rhodococcus sp. NCIMB9784and62%identity to P450smo from Rhodococcus sp. ECU0066, suggesting a natural fusion protein consisting of a FMN/[2Fe-2S] reductase partner, and monooxygenase domain.P450MO was successfully overexpressed in Escherichia coli (DE3), purified, and characterized for its spectroscopic and catalytic properties. The purified P450LaMO exhibited a characteristic Fe(II)CO complex with Soret peak at450nm. Type I (substrate) and type II (inhibitor) binding of p-chlorothioanisole and imzole were shown, with apparent dissociation constants of17.5μM and1.75μM. Steady-state reduction of cytochrome c was supported by either NADPH or NADH, with the Kcat of20.6min-1and12.9min-1, respectively. Kinetic study of NAD(P)H-dependent sulfoxidation ofp-chlorothioanisole also confirmed the slightly preference for NADPH. The optimum pH and temperature of purified P450LaMO were pH8.5and40℃. The half-lives of P450LaMO measured at20℃,30℃, and40℃were23h,15h, and2h, respectively. This suggests that higher temperatures will result in a rapid loss of activity.Substrates spectrum study of P450LaMO revealed that it was a versatile catalyst promoted the O-dealkylation, hydroxylation, olefin epoxidation and asymmetric sulfoxidation of a range of substituted aromatics. Such diverse activity is uncommon for wild-type CYPs, especially stereoselective hydroxylation at unactivated C-H bonds, which are ubiquitous and rather unreactive in organic molecules. P450LaMO could stereoselectively hydroxylate alkyl aromatic hydrocarbons, small bicyclic molecules, and even can regioselectively hydroxylate the terpenoid substrate β-ionone. Although, the P450-redox fusion enzymes of CYP116B family have not been crystallized which is one of the biggest obstacle to the extensively study. The self-sufficient nature of P450LaMO coupled with its newly uncovered broad scope highlights it as an ideal selection for directed evolution.