posted on 2023-12-14, 21:09authored byGustavo
J. Costa, Abel Egbemhenghe, Ruibin Liang
Unspecific peroxygenases (UPOs) are
emerging as promising biocatalysts
for selective oxyfunctionalization of unactivated C–H bonds.
However, their potential in large-scale synthesis is currently constrained
by suboptimal chemical selectivity. Improving the selectivity of UPOs
requires a deep understanding of the molecular basis of their catalysis.
Recent molecular simulations have sought to unravel UPO’s selectivity
and inform their design principles. However, most of these studies
focused on substrate-binding poses. Few researchers have investigated
how the reactivity of CpdI, the principal oxidizing intermediate in
the catalytic cycle, influences selectivity in a realistic protein
environment. Moreover, the influence of protein electrostatics on
the reaction kinetics of CpdI has also been largely overlooked. To
bridge this gap, we used multiscale simulations to interpret the regio-
and enantioselective hydroxylation of the n-heptane
substrate catalyzed by Agrocybe aegerita UPO (AaeUPO). We comprehensively characterized
the energetics and kinetics of the hydrogen atom-transfer (HAT) step,
initiated by CpdI, and the subsequent oxygen rebound step forming
the product. Notably, our approach involved both free energy and potential
energy evaluations in a quantum mechanics/molecular mechanics (QM/MM)
setting, mitigating the dependence of results on the choice of initial
conditions. These calculations illuminate the thermodynamics and kinetics
of the HAT and oxygen rebound steps. Our findings highlight that both
the conformational selection and the distinct chemical reactivity
of different substrate hydrogen atoms together dictate the regio-
and enantio-selectivity. Building on our previous study of CpdI’s
formation in AaeUPO, our results indicate that the
HAT step is the rate-limiting step in the overall catalytic cycle.
The subsequent oxygen rebound step is swift and retains the selectivity
determined by the HAT step. We also pinpointed several polar and charged
amino acid residues whose electrostatic potentials considerably influence
the reaction barrier of the HAT step. Notably, the Glu196 residue
is pivotal for both the CpdI’s formation and participation
in the HAT step. Our research offers in-depth insights into the catalytic
cycle of AaeUPO, which will be instrumental in the
rational design of UPOs with enhanced properties.