On the Mechanism and Specificity of Soluble, Quinoprotein Glucose Dehydrogenase in the Oxidation of Aldose Sugars

Olsthoorn, Arjen J. J.; Duine, Johannis A.

doi:10.1021/bi9808868

“…The first mechanism includes general base-catalyzed proton abstraction from the oxidizable hydroxyl group, followed by the formation of a covalent substrate-PQQ complex and product elimination (Fig. 2 A) (8)(9)(10)(11). The second possible reaction mechanism involves general basecatalyzed proton abstraction in concert with hydride transfer to PQQ and subsequent tautomerization to pyrroloquinoline quinone (PQQH 2 ) (Fig.…”

Section: Q Uinoproteins Form a Class Of Dehydrogenases Distinct From mentioning

confidence: 99%

“…The second possible reaction mechanism involves general basecatalyzed proton abstraction in concert with hydride transfer to PQQ and subsequent tautomerization to pyrroloquinoline quinone (PQQH 2 ) (Fig. 2B) (10)(11)(12). The substrate or hydride anion is normally considered to form an adduct at the PQQ C5 position (8-11, 13, 14), but Zheng and Bruice (12) have calculated that a mechanism involving hydride transfer from the substrate to the PQQ C4 atom may be energetically favorable.…”

Section: Q Uinoproteins Form a Class Of Dehydrogenases Distinct From mentioning

confidence: 99%

Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine: A covalent cofactor-inhibitor complex

Oubrie

¹

,

Rozeboom

²

,

Dijkstra

³

1999

Proc. Natl. Acad. Sci. U.S.A.

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Soluble glucose dehydrogenase (s-GDH) from the bacterium Acinetobacter calcoaceticus is a classical quinoprotein. It requires the cofactor pyrroloquinoline quinone (PQQ) to catalyze the oxidation of glucose to gluconolactone. The precise catalytic role of PQQ in s-GDH and several other PQQ-dependent enzymes has remained controversial because of the absence of comprehensive structural data. We have determined the crystal structure of a ternary complex of s-GDH with PQQ and methylhydrazine, a competitive inhibitor of the enzyme. This complex, refined at 1.5-Å resolution to an R factor of 16.7%, affords a detailed view of a cofactorbinding site of s-GDH. Moreover, it presents the first direct observation of covalent PQQ adduct in the active-site of a PQQ-dependent enzyme, thereby confirming previous evidence that the C5 carbonyl group of the cofactor is the most reactive moiety of PQQ. Quinoproteins form a class of dehydrogenases distinct from the NAD(P) ϩ -and flavin-dependent enzymes (1). They use one of four different quinone cofactors for the oxidation of a wide variety of compounds (2-5). The prototypical cofactor pyrroloquinoline quinone (PQQ; Fig. 1) is present in the bacterial quinoproteins methanol dehydrogenase (MDH) and glucose dehydrogenase (GDH) (2, 6, 7). On the basis of a variety of biochemical and kinetic data for MDH and the soluble GDH (s-GDH), two reaction mechanisms have been proposed for PQQ-containing enzymes (Fig. 2). The first mechanism includes general base-catalyzed proton abstraction from the oxidizable hydroxyl group, followed by the formation of a covalent substrate-PQQ complex and product elimination ( Fig. 2 A) (8-11). The second possible reaction mechanism involves general basecatalyzed proton abstraction in concert with hydride transfer to PQQ and subsequent tautomerization to pyrroloquinoline quinone (PQQH 2 ) ( Fig. 2B) (10-12). The substrate or hydride anion is normally considered to form an adduct at the PQQ C5 position (8-11, 13, 14), but Zheng and Bruice (12) have calculated that a mechanism involving hydride transfer from the substrate to the PQQ C4 atom may be energetically favorable. Thus, structural information is required to undisputedly resolve which of the two carbonyl groups of the ortho-quinone group of PQQ is most reactive in an enzymatic environment.s-GDH has long been thought to be exclusively present in the periplasmic space of the bacterium Acinetobacter calcoaceticus, but recently homologous sequences have been identified in the genomes of four other bacteria (15). s-GDH is a basic (pI ϭ 9.5) dimeric enzyme of identical subunits of 50 kDa each (454 residues after cleavage of a 24-residue signal peptide) (16, 17). The enzyme requires calcium for dimerization as well as for binding of PQQ (18). It oxidizes a broad range of aldose sugars to the corresponding lactones, with concomitant reduction of PQQ to PQQH 2 (Fig. 2) (19). Several artificial electron acceptors are able to reoxidize the cofactor (16). Stopped-flow kinetics indicated that the oxidation of glucose ...

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“…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)(16)(17). Accurate structural information will be valuable in distinguishing these two proposed mechanisms.…”

mentioning

confidence: 99%

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

Zheng

¹

,

Xia

²

,

Chen

³

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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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...

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“…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)(14)(15)(16). The second mechanism (Fig.…”

mentioning

confidence: 99%

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

Zheng¹

2001

Proceedings of the National Academy of Sciences

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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...

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On the Mechanism and Specificity of Soluble, Quinoprotein Glucose Dehydrogenase in the Oxidation of Aldose Sugars

Cited by 65 publications

References 27 publications

Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine: A covalent cofactor-inhibitor complex

Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine: A covalent cofactor-inhibitor complex

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

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

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