Elucidating the Enzymatic Mechanism of Dihydrocoumarin Degradation: Insight into the Functional Evolution of Methyl-Parathion Hydrolase from QM/MM and MM MD Simulations

Methyl-parathion hydrolase (MPH), which evolved from
dihydrocoumarin hydrolase, offers one of the most efficient enzymes
for the hydrolysis of methyl-parathion. Interestingly, the substrate
preference of MPH shifts from the methyl-parathion to the lactone
dihydrocoumarin (DHC) after its mutation of five specific residues
(R72L, L273F, L258H, T271I, and S193Δ, m5-MPH). Here, extensive
QM/MM calculations and MM MD simulations have been used to delve into
the structure–function relationship of MPH enzymes and plausible
mechanisms for the chemical and nonchemical steps, including the transportation
and binding of the substrate DHC to the active site, the hydrolysis
reaction, and the product release. The results reveal that the five
mutations remodel the active pocket and reposition DHC within the
active site, leading to stronger enzyme–substrate interactions.
The MM/GBSA-estimated binding free energies are about −20.7
kcal/mol for m5-MPH and −17.1 kcal/mol for wild-type MPH. Furthermore,
this conformational adjustment of the protein may facilitate the chemical
step of DHC hydrolysis and the product release, although there is
a certain influence on the substrate transport. The hydrolytic reaction
begins with the nucleophilic attack of the bridging OH– with the energy barriers of 22.0 and 18.0 kcal/mol for the wild-type
and m5-MPH enzymes, respectively, which is rate-determining for the
entire process. Unraveling these mechanistic intricacies may help
in the understanding of the natural evolution of enzymes for diverse
substrates and establish the enzyme structure–function relationship.