Binding of a physiological substrate causes large-scale conformational reorganization in cytochrome P450 51

作者：Tatiana Y. Hargrove, Z. Wawrzak, Paxtyn M. Fisher, Stella A. Child, W. David Nes, F. Peter Guengerich, Michael R. Waterman, Galina I. Lepesheva · 发表于：Journal of Biological Chemistry · 年份：2018 · DOI：10.1074/jbc.ra118.005850 · 被引用次数：23 · 研究领域：Pharmacogenetics and Drug Metabolism、Signaling Pathways in Disease、Trypanosoma species research and implications

Sterol 14α-demethylases (CYP51s) are phylogenetically the most conserved cytochromes P450, and their three-step reaction is crucial for biosynthesis of sterols and serves as a leading target for clinical and agricultural antifungal agents. The structures of several (bacterial, protozoan, fungal, and human) CYP51 orthologs, in both the ligand-free and inhibitor-bound forms, have been determined and have revealed striking similarity at the secondary and tertiary structural levels, despite having low sequence identity. Moreover, in contrast to many of the substrate-promiscuous, drug-metabolizing P450s, CYP51 structures do not display substantial rearrangements in their backbones upon binding of various inhibitory ligands, essentially representing a snapshot of the ligand-free sterol 14α-demethylase. Here, using the obtusifoliol-bound I105F variant of Trypanosoma cruzi CYP51, we report that formation of the catalytically competent complex with the physiological substrate triggers a large-scale conformational switch, dramatically reshaping the enzyme active site (3.5–6.0 Å movements in the FG arm, HI arm, and helix C) in the direction of catalysis. Notably, our X-ray structural analyses revealed that the substrate channel closes, the proton delivery route opens, and the topology and electrostatic potential of the proximal surface reorganize to favor interaction with the electron-donating flavoprotein partner, NADPH–cytochrome P450 reductase. Site-directed mutagenesis of the amino ...