Catalytic mechanism of quinoprotein methanol dehydrogenase: A theoretical and x-ray crystallographic investigation

Abstract

The catalytic mechanism of the reductive half reaction of the quinoprotein methanol dehydrogenase (MDH) is believed to proceed either through a hemiketal intermediate or by direct transfer of a hydride ion from the substrate methyl group to the cofactor, pyrroloquinoline quinone (PQQ). A crystal structure of the enzyme-substrate complex of a similar quinoprotein, glucose dehydrogenase, has recently been reported that strongly favors the hydride transfer mechanism in that enzyme. A theoretical analysis and an improved refinement of the 1.9-Å resolution crystal structure of MDH from Methylophilus methylotrophus W3A1 in the presence of methanol, reported earlier, indicates that the observed tetrahedral configuration of the C-5 atom of PQQ in that study represents the C-5-reduced form of the cofactor and lends support for a hydride transfer mechanism for MDH. Methanol and glucose dehydrogenases are two well studied quinoproteins that use 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) as a cofactor ( Fig. 1A; refs. 1-4). Both enzymes require a divalent cation such as Ca 2ϩ for catalytic activity. Crystallographic studies have been reported for both methanol dehydrogenase (MDH) and glucose dehydrogenase (5-12). These crystallographic investigations have not only provided detailed information concerning the PQQ binding site, but they have also established structural frameworks for mechanistic elucidation. Two plausible mechanisms have been proposed. The first one (Fig. 1B) involves a nucleophilic addition of the alcohol to the C-5 carbonyl followed by an intramolecular retro-ene reaction (13-16). The second mechanism (Fig. 1C) involves a general base initiated hydride transfer from the alcohol to the C-5 carbonyl carbon and subsequent tautomerization of the hydride transfer intermediate to the reduced PQQH2 (15-17). Accurate structural information will be valuable in distinguishing these two proposed mechanisms.Recently, a high-resolution structure of methanol dehydrogenase from Methylophilus methylotrophus W3A1 in a second crystal form has been reported (8). Surprisingly, even when planarity constraints were applied during the structural refinement, the refined structure displayed a C-5 center significantly distorted from planarity. Although the cofactor was assigned to be the semiquinone form of PQQ, this did not provide a satisfactory explanation as to why the C-5 center is tetrahedral, because earlier quantum mechanical studies had demonstrated that the tricyclic ring in the semiquinone form of PQQ is essentially planar in the presence or absence of Ca 2ϩ (17). The C-5 methanol adduct could give a tetrahedral C-5 center, but it is not compatible with the electron density map. The tautomeric form of the reduced PQQ, in which the pyrrole H-N hydrogen is moved to the C-5 carbon, does not seem to be plausible owning to a strong intramolecular hydrogen bond interaction between the pyrrole H-N and one of the C-9 carboxylate oxygen atoms. Thus, the observed tetrahedral C-5 remained an unresolved issue. However, if one loo...

Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization

“…(a) Structure of the PQQ prosthetic group. (b) Hydride transfer mechanism for methanol oxidation in PQQ-dependent MDH enzymes [49]. Asp297 is located in the large, catalytic a subunit.…”

Structure and Protein–Protein Interactions of Methanol Dehydrogenase from Methylococcus capsulatus (Bath)

“…In addition, the structure used in the cryoEM model is that of MDH from the methylotroph Methylophilus methylotrophus W3A1. 34 , 35 The structure of the cognate M. capsulatus (Bath) MDH has not been determined, hindering further consideration of the structural model. Moreover, the enhancement of pMMO propylene epoxidation activity by the presence of MDH could not be reconstituted by combining purified pMMO and MDH, and formation of a complex between purified pMMO and MDH has not been reported.…”

Mechanism of PQQ and CoQ10 overproduction in Methylobacterium as revealed by comparative genomic analysis between mutant and wild-type strains

“…The methanol dehydrogenase in methylotrophic bacteria is MXAF-type MDH, which is a quinone protein with PQQ as a prosthetic group (21). It serves as the vital enzyme for the methylotrophic bacteria, which facilitates methanol utilization to generate energy for bacterial growth (22).…”