Rationally Controlling Selective Steroid Hydroxylation via Scaffold Sampling of a P450 Family

Many steroids are important pharmaceutically active compounds,
while cytochrome P450 monooxygenases (CYPs) are attractive enzymes
for applications in steroidal drug synthesis. However, the catalytic
efficiency of existing P450s is not routinely high enough, as well
as the molecular basis for selectivity control is unclear, which severely
restrict their real applications. Here, a 16β steroid-hydroxylase
CYP109B4 from Bacillus sonorensis is
identified with excellent selectivity and activity. The crystallization
and structural analysis of CYP109B4 reveal potential three “hotspot”
residues (V84, V292, and S387) responsible for selectivity control.
Then, guided by the sequence–function relationships revealed
from the mutability landscape construction on the three residues,
focused rational iterative site-specific mutagenesis (FRISM) and limited
iterative saturation mutagenesis were performed, which provide variant
B4-M7 (L240V/S387F/V84L/V292S/I291T/M290F/F294I) with completely switched
regioselectivity from 16β to 15β. The subsequent computational
analysis uncovers insights into the substrate binding modes in CYP109B4
and its variants, which further confirms the critical role of the
“hotspot” residues for selectivity control. Finally,
the generality of conserved-“hotspots”-mediated selectivity
control is demonstrated by performing scaffold sampling between a
panel of CYP109B members. Overall, in addition to the present chemical
results, our study provides guidance in rationally designing more
excellent P450 biocatalysts for potential practical (industrial) applications.