Molecular Dynamics and QM/MM Calculations Predict the Substrate-Induced Gating of Cytochrome P450 BM3 and the Regio- and Stereoselectivity of Fatty Acid Hydroxylation

Theory predicts herein enzymatic
activity from scratch. We show
that molecular dynamics (MD) simulations and quantum-mechanical/molecular
mechanics (QM/MM) calculations of the fatty acid hydroxylase P450
BM3 predict the binding mechanism of the fatty acid substrate and
its enantio/regioselective hydroxylation by the active species of
the enzyme, Compound I. The MD simulations show that the substrate’s
entrance involves hydrogen-bonding interactions with Pro25, Glu43,
and Leu188, which induce a huge conformational rearrangement that
closes the substrate channel by pulling together the A helix and the
β1 sheet to the F/G loop. In turn, at the bottom
of the substrate’s channel, residue Phe87 controls the regioselectivity
by causing the substrate’s chain to curl up and juxtapose its
CH2 positions ω-1/ω-2/ω-3 to Compound
I while preventing access to the endmost position, ω-CH3. Phe87 also controls the stereoselectivity by the enantioselective
steric blocking of the pro-S C–H bond, thus
preferring R hydroxylation. Indeed, the MD simulations
of the mutant Phe87Ala predict predominant ω hydroxylation.
These findings, which go well beyond the X-ray structural data, demonstrate
the predictive power of theory and its insight, which can potentially
be used as a partner of experiment for eventual engineering of P450
BM3 with site-selective C–H functionalization capabilities.