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PubMed1995

The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens.

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

Alcohol Oxidoreductases↗
PubMed1995

The structure of the quinoprotein alcohol dehydrogenase of Acetobacter aceti modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

The 1.94 A structure of methanol dehydrogenase has been used to provide a model structure for part of a membrane quinohaemoprotein alcohol dehydrogenase. The basic superbarrel structure and the active-site region are retained, indicating essentially similar mechanisms of action, but there are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site.

Acetobacter↗
PubMed1995

Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens.

The structure of methanol dehydrogenase (MDH) at 0.194 nm (1.94 A) has been used to provide a model structure for part of a membrane quinoprotein glucose dehydrogenase (GDH). The basic superbarrel structure is retained, along with the tryptophan-docking motifs. The active-site regions are similar, but there are important differences, the most important being that GDH lacks the novel disulphide ring structure formed from adjacent cysteines in MDH; in GDH the equivalent region is occupied by His-262. Because of the overall similarities in the active-site region, the mechanism of action of GDH is likely to be similar to that of MDH. The differences in co-ordination to the cation and bonding to the pyrrolo-quinoline quinone (PQQ) in the active site may explain the relative ease of dissociation of the prosthetic group from the holo-GDH. There are considerable differences in the external loops, particularly those involved in formation of the shallow funnel leading to the active site, the configuration of which influences substrate specificity. The proposed model is consistent in many respects with previous proposals for the active-site structure based on the effects of chemical modification on binding of PQQ and enzymic activity.

Alcohol Oxidoreductases↗
PubMed1996

Characterization of a novel methanol dehydrogenase containing a Ba2+ ion at the active site.

The quinoprotein methanol dehydrogenase (MDH) contains a Ca2+ ion at the active site. Ca(2-)-free enzyme (from a processing mutant) was used to obtain enzyme containing Sr2+ or Ba2+, the Ba(2+)-MDH being the first enzyme to be described in which a Ba2+ ion functions at the active site. The activation energy for oxidation of methanol by Ba(2+)-MDH is less than half that of the reaction catalysed by Ca(2+)-MDH (a difference of 21.4 kJ/mol), and the Vmax value is 2-fold higher. The affinities of Ba(2+)-MDH for substrate and activator are very much less than those of Ca(2+)-MDH; the Km for methanol is 3.5 mM (compared with 3 microM) and the KA for ammonia is 52 mM (compared with 2 mM). The different activity of Ba(2+)-MDH is probably due to a change in the conformation of the active site, leading to a decrease in the free energy of substrate binding and hence a decrease in activation energy. The kinetic model for Ba(2+)-MDH with respect to substrate and activator is consistent with previous models for Ca(2+)-MDH. The pronounced deuterium isotope effect (6.0-7.6) is influenced by ammonia, and is consistent with activation of the pyrroloquinoline quinone reduction step by ammonia. Because of its low affinity for substrates, it is possible to prepare the oxidized form of Ba(2+)-MDH. No spectral intermediates could be detected during reduction by added substrate, and so it is not possible to distinguish between those mechanisms involving covalent substrate addition and those involving only hydride transfer.

Alcohol Oxidoreductases↗
PubMed1996

Quinoprotein-catalysed reactions.

This review is concerned with the structure and function of the quinoprotein enzymes, sometimes called quinoenzymes. These have prosthetic groups containing quinones, the name thus being analogous to the flavoproteins containing flavin prosthetic groups. Pyrrolo-quinoline quinone (PQQ) is non-covalently attached, whereas tryptophan tryptophylquinone (TTQ), topaquinone (TPQ) and lysine tyrosylquinone (LTQ) are derived from amino acid residues in the backbone of the enzymes. The mechanisms of the quinoproteins are reviewed and related to their recently determined three-dimensional structures. As expected, the quinone structures in the prosthetic groups play important roles in the mechanisms. A second common feature is the presence of a catalytic base (aspartate) at the active site which initiates the reactions by abstracting a proton from the substrate, and it is likely to be involved in multiple reactions in the mechanism. A third common feature of these enzymes is that the first part of the reaction produces a reduced prosthetic group; this part of the mechanism is fairly well understood. This is followed by an oxidative phase involving electron transfer reactions which remain poorly understood. In both types of dehydrogenase (containing PQQ and TTQ), electrons must pass from the reduced prosthetic group to redox centres in a second recipient protein (or protein domain), whereas in amine oxidases (containing TPQ or LTQ), electrons must be transferred to molecular oxygen by way of a redox-active copper ion in the protein.

Alcohol Oxidoreductases↗
PubMed2002

The essential nutrient pyrroloquinoline quinone may act as a neuroprotectant by suppressing peroxynitrite formation.

Pyrroloquinoline quinone (PQQ) is a redox active essential nutrient that can generate or scavenge superoxide depending on its microenvironment. PQQ has been shown previously to be neuroprotective in a rodent stroke model. Here we test whether PQQ interacts with reactive nitrogen species, known to be involved in the pathogenesis of stroke. Using rat forebrain neurons in culture, we determined that the toxicity of SIN-1 was mediated by peroxynitrite and that PQQ could block this toxic action. However, PQQ could not block the toxicity of peroxynitrite itself. Both SIN-1 and peroxynitrite caused ATP depletion, but only SIN-1 evoked ATP depletion was blocked by PQQ. In a cell-free system, PQQ blocked nitration of bovine serum albumin produced by SIN-1, but potentiated peroxynitrite-induced nitration. PQQ was unable to block ATP depletion and cell death induced by NO. donors (DEA/NO, DPT/NO and DETA/NO), indicating that it does not directly interact with nitric oxide, and suggesting that it acts as a superoxide scavenger. PQQ significantly potentiated cGMP accumulation evoked by SIN-1, similar to the effect of superoxide dismutase (SOD). However, unlike SOD, which potentiated neurotoxicity induced by SIN-1, PQQ blocked its toxicity, arguing against the possibility that PQQ functions simply as a SOD mimetic. Indeed, substantially less H2O2 was produced by the incubation of SIN-1 with PQQ, when compared to SOD. These results suggest that PQQ scavenges superoxide without forming toxic levels of H2O2. Therefore, the protective effect of PQQ on stroke might be due, at least in part, to the suppression of peroxynitrite formation.

Animals↗
PubMed1997

Conformation of coenzyme pyrroloquinoline quinone and role of Ca2+ in the catalytic mechanism of quinoprotein methanol dehydrogenase.

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-CO2-general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.

Alcohol Oxidoreductases↗
PubMed2001

Pyrroloquinoline quinone (PQQ) and quinoprotein enzymes.

This review summarises the characteristics, identification, and measurement of pyrroloquinoline quinone, the prosthetic group of bacterial quinoprotein dehydrogenases whose structures, mechanisms, and electron transport functions are described in detail. Type I alcohol dehydrogenase includes the "classic" methanol dehydrogenase; its x-ray structure and mechanism are discussed in detail. It is likely that its mechanism involves a direct hydride transfer rather than a mechanism involving a covalent adduct. The x-ray structure of a closely related ethanol dehydrogenase is also described. The type II alcohol dehydrogenase is a soluble quinohaemoprotein, having a C-terminal extension containing haem C, which provides an excellent opportunity for the study of intraprotein electron transfer processes. The type III alcohol dehydrogenase is similar but it has two additional subunits (one of which is a multihaem cytochrome c) bound in an unusual way to the periplasmic membrane. One type of glucose dehydrogenase is a soluble quinoprotein whose role in energy transduction is uncertain. Its x-ray structure (in the presence and absence of substrate) is described together with the detailed mechanism, which also involves a direct hydride transfer. The more widely distributed glucose dehydrogenases are integral membrane proteins, bound to the membrane by transmembrane helices at the N-terminus.

Alcohol Oxidoreductases↗
PubMed1999

Action of pyrroloquinolinequinol as an antioxidant against lipid peroxidation in solution.

The activities of pyrroloquinoline quinone (PQQ), a coenzyme of methanol dehydrogenase and amine oxidase, and its reduced form pyrroloquinoline quinol (PQQH2) as an antioxidant have been measured in solution. PQQH2 was stable in the absence of oxygen but rapidly auto-oxidized to PQQ in the presence of oxygen in water. PQQH2 was stable in an aprotic solvent such as acetonitrile, even in air. PQQ did not exert appreciable antioxidant activity, whereas PQQH2 exerted higher reactivity than alpha-tocopherol toward galvinoxyl radical and peroxyl radical. PQQH2 acted as a potent antioxidant against the oxidation of methyl linoleate in acetonitrile induced by azo compound and produced a clear induction period, from which the apparent stoichiometric number was obtained as 1.1. PQQH2 reduced the alpha-tocopheroxyl radical and spared alpha-tocopherol in the oxidation of methyl linoleate. These results suggest that PQQH2 may act as a potent antioxidant, particularly in combination with alpha-tocopherol.

Animals↗
PubMed1994

Dietary pyrroloquinoline quinone: growth and immune response in BALB/c mice.

Pyrroloquinoline quinone (PQQ) is proposed as a nutritionally important growth factor, and we provide evidence that PQQ improves reproduction performance in BALB/c mice and stimulates neonatal growth. In the first experiment, weanling female BALB/c mice were adapted to a chemically-defined diet containing 0, 100, 200, 300, 1000, or 5000 ng PQQ/g of diet. The mice were bred and their reproductive performance and surviving offspring were assessed for 20-wk. Reproductive outcome was markedly compromised for the groups most deprived of PQQ. Supplemented groups (> or = 1000 ng PQQ/g diet) had 8 pups/litter compared with 4-5 pups/litter in the PQQ-deprived groups (< or = 300 ng PQQ/g diet). Of the pups surviving to weaning, 8 of 10 survived when PQQ was added to the diet (> or = 300 ng PQQ/g diet) compared with 4 of 10 in the PQQ/deficient group. The apparent requirement for PQQ for optimal growth of surviving neonates was estimated to be > or = 300 ng PQQ/g of diet. Moreover, splenic cell response to the mitogens concanavalin A and lipopolysaccharide, appeared related to PQQ intake. In a second experiment, female BALB/c mice were fed diets containing PQQ added at 0 or 1000 ng/g of diet, and interleukin 1 and 2 production were assessed. In particular, levels of interleukin 2, an autocrine and paracrine growth factor, were reduced in mice fed the deficient diet at a time when T-cell proliferation occurs in neonates. Results suggest that PQQ or similar compounds may play nutritionally important roles at critical stages in development.

Animals↗
PubMed2006

Pyrroloquinoline quinone modulates mitochondrial quantity and function in mice.

When pyrroloquinoline quinone (PQQ) is added to an amino acid-based, but otherwise nutritionally complete basal diet, it improves growth-related variables in young mice. We examined PQQ and mitochondrial function based on observations that PQQ deficiency results in elevated plasma glucose concentrations in young mice, and PQQ addition stimulates mitochondrial complex 1 activity in vitro. PQQ-deficient weanling mice had a 20-30% reduction in the relative amount of mitochondria in liver; lower respiratory control ratios, and lower respiratory quotients than PQQ-supplemented mice (2 mg PQQ/kg diet). In mice from dams fed a conventional laboratory diet, but switched at weaning to the basal diet, plasma glucose, Ala, Gly, and Ser concentrations were elevated at 4 wk (PQQ- vs. PQQ+), but not at 8 wk. The relative mitochondrial content (ratio of mtDNA to nuclear DNA) also tended (P<0.18) to be lower (PQQ- vs. PQQ+) at 4 wk, but not at 8 wk. PQQ also counters the mitochondrial complex 1 inhibitor, diphenylene iodonium (DPI). Mice were gavaged with 0, 0.4, or 4 microg PQQ/g body weight (BW) daily for 14 d. At each PQQ level, DPI was injected (i.p.) at 0, 0.4, 0.8, or 1.6 microg DPI/g BW. The PQQ-deficient mice exposed to 0.4 or 4.0 microg DPI/g lost weight and had lower plasma glucose levels than PQQ-supplemented mice (P<0.05). In addition, fibroblasts took up (3)H-PQQ added to cell cultures, and cultured hepatocytes maintained mitochondrial PQQ concentrations similar to those observed in vivo. Collectively, these results indicate that dietary PQQ can influence mitochondrial amount and function, particularly in perinatal and weanling mice.

Amino Acids↗
PubMed1995

Production and application of monoclonal antibodies specific to pyrroloquinoline quinone.

We produced five monoclonal antibodies (mAbs 1, 2, 6, 7, and 9) that are specific to pyrroloquinoline quinone (PQQ). PQQ-conjugated hemocyanin was used for the immunization of mice and the hybridomas were selected using PQQ-conjugated BSA in an enzyme-linked immunosorbent assay. MAbs 2 and 9 were of the IgG1 isotype. Both could recognize free PQQ, the former probably at the o-quinone and the latter at the opposite side of the molecule. They did not bind with trihydroxyphenylalanine, dihydroxyphenylalanine, 1,2,4-trihydroxybenzene, ascorbic acid, riboflavin, or menadione. In contrast to the IgGs, mAbs 1, 6, and 7 (IgMs) did not bind with free PQQ. Using mAb 2, a competitive enzyme-linked immunosorbent assay was developed, which enabled us to determine 50 nM-1 microM free PQQ. Furthermore, we analyzed the covalently bound prosthetic groups of two quinoproteins (amine oxidase from Aspergillus niger and amine dehydrogenase from Pseudomonas putida) by Western analysis using these mAbs. However, the results was negative, indicating that the prosthetic groups are not PQQ.

Animals↗
PubMed1988

Localization of a pyrroloquinoline quinone biosynthesis gene near the methanol dehydrogenase structural gene in Methylobacterium organophilum DSM 760.

A partial Sau3AI genomic bank of Methylobacterium organophilum DSM 760 was constructed in the cosmid pSUP106 and moxF, the structural gene for methanol dehydrogenase, was isolated. In M. organophilum, pSUP106 behaves as a suicide plasmid. This property was used to insert Tn5 into the bacterial chromosome, in the vicinity of moxF, by marker exchange. Mobilization of the Tn5-labelled chromosomal region by a broad-host-range plasmid, pJB3J1 (an R68-45 derivative), allowed the selection of several large R' hybrid plasmids in Escherichia coli HB101. Most of them were able to complement both mutants of the moxF region and mutant MTM1, the first mutant of the pyrroloquinoline quinone (PQQ) biosynthesis pathway in M. organophilum. The gene involved, pqqA, was subcloned and localized.

Alcohol Oxidoreductases↗
PubMed1991

Mutants of Escherichia coli producing pyrroloquinoline quinone.

In glucose minimal medium a PTS- strain of Escherichia coli [delta (ptsH ptsI crr)] could grow slowly (doubling time, d = 10 h). When the population reached 5 x 10(6) to 2 x 10(7) cells ml-1, mutants growing rapidly (d = 1.5 h) appeared and rapidly outgrew the initial population. These mutants (EF mutants) do not use a constitutive galactose permease for glucose translocation. They synthesize sufficient pyrroloquinoline quinone (PQQ) to yield a specific activity of glucose dehydrogenase (GDH) equivalent to that found in the parent strain grown in glucose minimal medium supplemented with 1 nM-PQQ. Membrane preparations containing an active GDH oxidized glucose to gluconic acid, which was also present in the culture supernatant of EF strains in glucose minimal medium. Glucose utilization is the only phenotypic trait distinguishing EF mutants from the parent strain. Glucose utilization by EF mutants was strictly aerobic as expected from a PQQ-dependent catabolism. The regulation of PQQ production by E. coli is discussed.

Aerobiosis↗
PubMed1997

Sequence analysis of pqq genes required for biosynthesis of pyrroloquinoline quinone in Methylobacterium extorquens AM1 and the purification of a biosynthetic intermediate.

Methylobacterium extorquens AM1 produces pyrroloquinoline quinone (PQQ), the prosthetic group of methanol dehydrogenase. Two genes clusters have been shown to be required for PQQ biosynthesis in this micro-organism and complementation analysis has identified seven pqq genes, pqqDGCBA and pqqEF. The DNA sequence of pqqDGC' was reported previously. This paper reports the sequence of the genomic region corresponding to pqqC'BA. For consistency, the nomenclature of pqq genes in Klebsiella pneumoniae will be followed. The new nomenclature for pqq genes of M. extorquens AM1 is pqqABCDE and pqqFG. In the genomic region sequenced in this study, two open reading frames were found. One of these encodes pqqE, which showed high identity to analogous pqq genes in other bacteria. PqqE also showed identity to MoaA and NifB in the N-terminal region, where a conserved CxxxCxYC sequence was identified. The sequence of the second open reading frame covered both the pqqC and pqqD regions, suggesting that both functions were encoded by this gene. It is proposed to designate this gene pqqC/D. The deduced amino acid sequence of the pqqC/D products showed identity to PqqC of K. pneumoniae and Pqql of Acinetobacter calcoaceticus in the N-terminal region, and to PqqD of K. pneumoniae and Pqql of A. calcoaceticus in the C-terminal region. A fragment of M. extorquens AM1 DNA containing only pqqC/D produced a protein of 42 kDa in Escherichia coli, which corresponds to the size of the deduced amino acid sequence of PqqC/D, confirming the absence of a separate pqqD. This genomic region complemented the growth of pqqC mutants of M. extorquens AM1 and Methylobacterium organophilum DSM 760 on methanol. As previously reported for pqq genes of K. pneumoniae, a pqqC mutant of M. extorquens AM1 produced an intermediate of PQQ biosynthesis, which was converted to PQQ by incubation with a crude extract from E.coli cells expressing PqqC/D. The intermediate was found in both crude extract and culture supernatant, and it was purified from the crude extract. The PqqC/D enzyme reaction appeared to require molecular oxygen and reduced nicotinamide adenine dinucleotides.

Amino Acid Sequence↗