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Binding of distinct substrate conformations enables hydroxylation of remote sites in thaxtomin D by cytochrome P450 TxtC

DOI: 10.1021/jacs.8b08864 DOI Help

Authors: Lona M. Alkhalaf (University of Warwick) , Sarah Barry (University of Warwick) , Dean Rea (University of Warwick) , Angelo Gallo (University of Warwick) , Daniel Griffiths (University of Warwick) , Jozef Lewandowski (University of Warwick) , Vilmos Fulop (University of Warwick) , Gregory L. Challis (University of Warwick; Monash University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of The American Chemical Society

State: Published (Approved)
Published: December 2018
Diamond Proposal Number(s): 8359

Abstract: Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation and natural product biosynthesis. Here we report biochemical, structural and theoretical studies of TxtC, an unusual bifunctional CYP in-volved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites with-in the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the α-carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionaliza-tion, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen com-plex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the π-face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally-distinct binding mode in which the α-hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the α-hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohydroxylated intermediate.

Subject Areas: Chemistry


Instruments: I24-Microfocus Macromolecular Crystallography