Mechanisms of ammonia activation and ammonium ion inhibition of quinoprotein methanol dehydrogenase: A computational approach

Abstract

The mechanism of methanol oxidation by quinoprotein methanol dehydrogenase (MDH⅐PQQ) in combination with methanol (MDH⅐PQQ⅐methanol) involves Glu-171OCO Methanol dehydrogenase (MDH) oxidoreductase is a soluble periplasmic quinoprotein important in oxidizing methanol to formaldehyde during growth of bacteria on methane (1-5). Pyrroloquinoline quinone (PQQ) or methoxatin (old name) is the noncovalently bound prosthetic group of MDH (Fig. 1a). The enzyme catalyzes methanol oxidation with PQQ reduction to hydroquinone (PQQH 2 ) and release of formaldehyde. This reaction is followed by two sequential single-electron transfers to the cytochrome c L , during which PQQH 2 is oxidized back to the quinone. In vitro, the oxidation of methanol occurs at pH 9 with phenazine methosulphate as an artificial electron acceptor. The rate-limiting step is the oxidation of substrate. Ammonia and methylamine are activators (6-8), but ammonium ion and high concentrations of ammonia act as inhibitors (8-10). Ortho-quinones react easily with primary amines to form carbinolamines, which are in equilibrium with the corresponding imines (11). Imines readily undergo nucleophilic addition by specific and general acid catalysis.Harris and Davidson (9) reported that a very small but certain spectral perturbation occurs on addition of NH 3 to an aqueous solution of PQQ, which has been associated with the formation of an iminoquinone adduct. This spectral feature was not seen on addition of NH 3 to a solution of MDH⅐PQQ (8). Thus, it was concluded that the PQQ moiety of MDH⅐PQQ does not react with NH 3 at low concentrations to form an imine (9). It has occurred to us that there are alternate explanations for the latter observation.Molecular dynamics (MD) simulations provide proper alignment of the active site residues in the enzyme-substrate and enzyme-intermediate complexes.Comparison of the x-ray coordinates of MDH intermediate formed during methanol oxidation and the ab initio structure of hydride adduct to the C5 of PQQ (12) established a mechanism (3), which involves the hydride transfer from methanol to PQQ to provide PQQH 2 (Fig. 2). This mechanism has been elaborated by MD simulation studies of MDH (13-15) and soluble glucose dehydrogenase (sGDH) (16) reactions. Further, the x-ray structure of sGDH⅐PQQ⅐glucose is best explained by hydride transfer in the oxidation of glucose by sGDH (17). In the present study, we examined the obvious means by which NH 3 ͞NH 4 ϩ can influence the rate of oxidation of methanol by probing the active site of MD structures of MDH⅐PQQ⅐methanol and the intermediate complex MDH⅐PQQH in the presence of NH 3 and also NH 4 ϩ . In addition, MD structures of the / ‫گ‬ C5ANH and / ‫گ‬ C5ANH 2 ϩ imine derivatives of MDH⅐PQQ⅐methanol have been investigated. MethodsThe partial atomic charges of PQQ, PQQ(NH), and PQQ(NH 2 ϩ ) (Fig. 1) were obtained by means of quantum chemical calculations by using GAUSSIAN 98 (18). In this procedure, Ca 2ϩ ion was included at an appropriate coordinating distance to the N6, O7A, and O5͞...

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.…”

“…3) of the PQQ methanol dehydrogenase (MDH⅐PQQ⅐MeOH⅐Wat1) complex in the water solvent was obtained from the final structure of 3-ns MD simulations (6,7,9). The ground state was obtained by QM/MM energy-minimizing the final structure from our previous MD simulation (6,7,9) for MDH⅐PQQ⅐MeOH⅐Wat1 complex by Adopted Basis Newton-Raphson (ABNR) method until the gradient was Ͻ0.01 kcal/(mol⅐Å). The similar procedure was used to obtain the QM/MM starting structure from the snapshot (1.2 ns) of 3-ns MD simulations for MDH⅐PQQ⅐MeOH model.…”

“…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.…”

“…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).…”