Substrate-induced conformational change in cytochrome P450 OleP

The regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analogue 6-deoxyerythronolide B (6DEB) crystallized in two forms, one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves a slow event of conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is likely bound to open OleP. Data herein discussed provide structural snapshots of key pre-catalytic steps of the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.