In Silico Studies of the Mechanism of Methanol Oxidation by Quinoprotein Methanol Dehydrogenase

Abstract

The mechanism of bacterial methanol dehydrogenase involves hydride equivalent transfer from substrate to the ortho-quinone PQQ to provide a C5-reduced intermediate that subsequently rearranges to the hydroquinone PQQH(2). We have studied the PQQ reduction by molecular dynamic (MD) simulations in aqueous solution. Among the five simulated structures, either Asp297 or Glu171 or both are ionized. Reasonable structures are obtained only when both carboxyl groups are ionized. This is not unexpected since the kinetic pH optimum is 9.0. In the structure of the enzyme.PQQ.HOCH(3) complex, the hydrogen bonded Glu171-CO(2)(-).H-OCH(3) is in a position to act as a general base catalyst for hydride equivalent transfer to C5 of PQQ. We thus suggest that Glu171 plays the role of general base catalyst in PQQ reduction rather than Asp297 as previously suggested. The reduction is assisted by Arg324, which hydrogen bonds to the ortho-quinone moiety of PQQ. The rearrangement of the C5-reduced intermediate to provide hydroquinone PQQH(2) is also assisted by proton abstraction by Glu171-CO(2)(-) and the continuous hydrogen bonding of Arg324 throughout the entire reaction. These features as well as the mapping of the channel for substrate and water into the active site entrance are the observations of major importance.

Mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

“…Computational findings based on the present QM/MM computations and MD simulations, as well as our previous MD simulations (6,7,9), lead to the conclusion that only Glu-171-CO 2 Ϫ could be the general base catalyst for the oxidation of CH 3 OH 3 CH 2 O.…”

“…Based on the procedures described in our previous study (9), stochastic boundary MD was carried out on [1A]…”

“…1-4). More recently, our attention has been directed to the employment of computational methods, particularly in the mechanism of the oxidation of methanol and glucose by the appropriate quinoprotein enzymes (5)(6)(7)(8)(9)(10)(11).…”

“…During 3-ns molecular dynamics (MD) simulations (6,7,9) with MeOH at the active site, it was found that MeOH became hydrogen bonded to Glu-171-CO 2 Ϫ , with the C-H of MeOH adjacent to the quinone C5 of PQQ and Wat1 hydrogen bonded to Asp-297-CO 2 Ϫ . This MD derived structure is not in accord with the following: (i) the suggestion that Asp-297-CO 2 Ϫ serves as the general-base catalyst (14)(15)(16)(17); and (ii) that the oxidation takes place, not by hydride transfer, but by an addition elimination reaction (18,19) (Scheme 1, in which the general base may be Glu-171-CO 2 Ϫ or Asp-297-CO 2 Ϫ ).…”

“…This MD derived structure is not in accord with the following: (i) the suggestion that Asp-297-CO 2 Ϫ serves as the general-base catalyst (14)(15)(16)(17); and (ii) that the oxidation takes place, not by hydride transfer, but by an addition elimination reaction (18,19) (Scheme 1, in which the general base may be Glu-171-CO 2 Ϫ or Asp-297-CO 2 Ϫ ). The MD studies (6,7,9) showed that Asp-297-CO 2 Ϫ is not in position to act as a generalbase catalyst for the oxidation of MeOH by MDH. The mechanism of Scheme 2 was proposed.…”