Substrate‐induced conformational change in cytochrome P450 OleP

Abstract

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 analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.-Parisi, G., Montemiglio, L. C., Giuffrè, A., Macone, A., Scaglione, A., Cerutti, G., Exertier, C., Savino, C., Vallone, B. Substrate-induced conformational change in cytochrome P450 OleP.

Dissecting the Cytochrome P450 OleP Substrate Specificity: Evidence for a Preferential Substrate

“…For OleP–DEO LS and OleP–DEO HS , initial phases were calculated by molecular replacement using MOLREP [ 25 ] from the CCP4 package (version 7.0). The coordinates of a single open monomer of OleP in complex with 6DEB in low salt conditions (pdb 5MNV) were used as model for OleP–DEO LS [ 20 ]; the atomic model of a single closed monomer of OleP-6DEB in high salt conditions (pdb 5MNS) was used as a template for OleP–DEO HS [ 20 ]. Thanks to space group isomorphism, the atomic models of OleP–DEO-rhamnose and of OleP–6DEB–rhamnose were determined by the Fourier synthesis method using respectively as templates the atomic coordinates of OleP–DEO HS and OleP-6DEB (PDB code 5MNS, [ 20 ]).…”

“…The recent availability of the OleP three-dimensional structure suggested that the active site of the enzyme could host both substrates [ 19 ]. Structural and kinetic studies performed with the aglycone substrate analog 6-deoxyerythronolide B (6DEB), from S. erythraeus , that only differs from DEO for the presence of an ethyl moiety at carbon 13 instead of a methyl unit ( Figure 1 B), revealed that substrate recognition in OleP is regulated by a complex mechanism that involves a large open-to-closed structural transition triggered by substrate binding, allowing the enzyme to adopt the catalytically competent conformation [ 20 ]. Notably, in the presence of 6DEB, only a minor population of the enzyme closes, showing that binding of this substrate does not fully favor the open-to-closed transition.…”

Conformational selection dominates binding of steroids to human cytochrome P450 17A1

“…The well-studied bacterial model P450 cam (P450 101A1) has been shown to exist in both open and closed states, and the relevance of these in different steps in the catalytic cycle is a subject of current interest (5)(6)(7)(8)(9). Another bacterial P450, OleP, has been recently reported to show multiple conformations in the presence of a substrate analog (10). Mammalian P450s have also been found to exist in open, closed, and "mixed" conformational states, at least in crystal structures (11)(12)(13)(14)(15)(16)(17).…”

Highly selective bile acid hydroxylation by the multifunctional bacterial P450 monooxygenase CYP107D1 (OleP)

“…Consequently, the space time yield of MDCA formation is 0.042 mM h -1 . Previously CYP107D1 was only described for its involvement in the synthesis of oleandomycin (Montemiglio et al 2016;Parisi et al 2018) and forming the 6b-, 17b-, 12b-, and 15b-hydroxylated derivatives of testosterone (Agematu et al 2006). To the best of our knowledge, the selective 6b-hydroxylation of LCA by CYP107D1 (OleP) had not been described.…”

“…The P450 monooxygenase CYP107D1, also known as OleP, is part of the oleandomycin synthesis pathway of Streptomyces antibioticus (Rodriguez et al 1995;Shah et al 2000). In this pathway it selectively introduces an epoxide functionality in a macrolide (Montemiglio et al 2016;Parisi et al 2018). More importantly, CYP107D1 has been reported to unspecifically hydroxylate testosterone (Agematu 2006).…”