2018_EJRNL_Lona_M__Alkhalaf_1.pdf
Terbatas Latifa Noor
» ITB
Terbatas Latifa Noor
» ITB
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 involved in the biosynthesis of the
EPA-approved herbicide thaxtomin A. TxtC was shown to
hydroxylate two remote sites within the Phe residue of its
diketopiperazine substrate thaxtomin D. The reactions follow
a preferred order, with hydroxylation of the ?-carbon
preceding functionalization of the phenyl group. To illuminate
the molecular basis for remote site functionalization, 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 complex into the
reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site,
with the ?-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 ?-hydrogen of the Phe residue
is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the ?-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.