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

Abstract

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 ...

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

“…This would eliminate the necessity to explain the tetrahedral C-5 center. The immediate implication of this hypothesis is that methanol dehydrogenase follows a hydride transfer mechanism, which is in agreement with the hydride transfer mechanism for glucose dehydrogenase demonstrated recently by Dijkstra and coworkers (11,12,22).…”

“…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)(6)(7)(8)(9)(10)(11)(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.…”

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation

“…The role of Ca 2+ may also indicate a more specific role. PQQ requiring enzymes such as methanol dehydrogenase and soluble glucose dehydrogenase (Oubrie et al 1999) require Ca 2+ for binding of PQQ and stabilization of the PQQ semiquinone form (Sato et al 2001). The effect of Ca 2+ on Mn(II) oxidation is consistent with catalysis by a PQQ requiring enzyme.…”

“…Surprisingly, o-phenanthroline, a specific copper chelator and known inhibitor of Mn(II) oxidation in other species, only partially inhibited Mn(II) oxidation at high concentrations-much higher than was required to inhibit Mn(II) oxidation in partially purified preparations of Pseudomonas putida GB-1 (Okazaki et al 1997). Hydrazines covalently bind to PQQ and are known inhibitors of PQQ utilizing enzymes (van der Meer et al 1987;Oubrie et al 1999). Phenylhydrazine inhibited Mn(II) oxidation, and was a very potent inhibitor (10 µM completely inhibited the reaction) when additional PQQ was omitted from the assay.…”

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes

“…The oxidised form of PQQ can be converted into the reduced form PQQH2 by the transfer of 2 electrons and two protons from the substrate molecule. [19][20][21] There are two types of PQQ-GDH enzymes that can be considered for biosensor design. One is intracellular and soluble (sPQQ-GDH) whereas the other molecule is insoluble and firmly bound to the outer surface of the cytoplasmic membrane (mPQQ-GDH).…”