Conformational Gating in CYP154C2: Gln230-Mediated Substrate Recognition and Catalytic Switching Revealed by Structural Dynamics.

Abstract

Previously, we reported that CYP154C2 from Streptomyces avermitilis is capable of catalyzing the 2α-hydroxylation of the two model substrates, testosterone (TES) and androstenedione (ASD), and resolved the closed structures of both the substrate-free form and the TES-bound form. In this study, we extend these findings by determining the open-conformation structures of the substrate-free and ASD-bound forms-a rare achievement among bacterial P450s. Structural analyses revealed coordinated conformational shifts in the FG helices, HI helices, and BC loop during open-to-closed transitions. Despite divergent overall conformations, both substrates positioned their C2 atoms near the heme iron, aligning for 2α-hydroxylation. Mutagenesis studies established Gln230's pivotal role in substrate recognition and catalytic activation. High-resolution crystallography (1.97 Å) of the Q230A mutant revealed PEG-occupied catalytic pockets (indicating complete loss of TES binding) while maintaining the open conformation. These results provide atomic-level evidence that Gln230 coordinates both substrate-driven conformational gating and catalytic site optimization.