Molecular Basis of Regioselective Bridgehead C( <i>sp</i> ³ )–H Bond Hydroxylation by P450 Peroxygenase in Bicyclomycin Biosynthesis

Achieving highly regioselective C–H bond functionalization at the bridgehead carbon of a single molecule is a fascinating, yet formidable challenge. BcmD, a P450 enzyme, catalyzes the regioselective hydroxylation at the sp3-hybridized bridgehead carbon of the bicyclo[4.2.2]piperazinedione ring scaffold during bicyclomycin biosynthesis. Herein, we provide mechanistic details of BcmD to understand its substrate binding and the origin of its regioselectivity. The high-resolution structure of SoBcmD from Streptomyces ossamyceticus in complex with its natural substrate, together with site-directed mutagenesis and computational simulations, reveals that the precise substrate positioning, enabled by specific hydrogen-bonding and hydrophobic interactions with crucial residues, determines the regioselectivity. Intriguingly, we identify SoBcmD as a peroxygenase that could utilize H2O2 as an oxidant to catalyze regioselective hydroxylation. Mechanistic analysis indicates that H2O2 activation in SoBcmD involves a homolytic cleavage process, mediated by a hydrogen-bonding network comprising residues T277, A273, and the substrate. Furthermore, structure-based channel analysis and mutagenesis demonstrate that the I157V mutant exhibits significantly enhanced peroxygenase activity. These findings advance our understanding of regioselectivity control in BcmD and provide valuable guidance for engineering P450 peroxygenases for the C–H bond activation at the chemically challenging bridgehead carbon sites.