Evidence for PQQ as cofactor in 3,4-dihydroxyphenylalanine (dopa) decarboxylase of pig kidney

Pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase (EC 4.1.1.28) was purified to homogeneity. Treatment of the enzyme with phenylhydrazine (PH) according to a procedure developed for analysis of quinoproteins gave products which were identified as the hydrazone of pyridoxal phosphate (PLP) and the C(5)-hydrazone of pyrroloquinoline quinone (PQQ). This method failed, however, in quantifying the amounts of cofactor. Direct hydrolysis of the enzyme by refluxing with hexanol and concentrated HCl led to detachment of PQQ from the protein in a quantity of 1 PQQ per enzyme molecule. In view of the reactivity of PQQ towards amines and amino acids, we postulate that it participates as a covalently bound cofactor in the catalytic cycle of the enzyme, in interplay with PLP. Since several other enzymes have been reported to show the atypical behaviour of dopa decarboxylase, it seems that the PLP-containing group of enzymes can be subdivided into pyridoxoproteins and pyridoxo-quinoproteins.     

Escherichia coli PQQ-containing quinoprotein glucose dehydrogenase: its structure comparison with other quinoproteins

Membrane-bound glucose dehydrogenase (mGDH) in Escherichia coli is one of the pivotal pyrroloquinoline quinone (PQQ)-containing quinoproteins coupled with the respiratory chain in the periplasmic oxidation of alcohols and sugars in Gram-negative bacteria. We compared mGDH with other PQQ-dependent quinoproteins in molecular structure and attempted to trace their evolutionary process. We also review the role of residues crucial for the catalytic reaction or for interacting with PQQ and discuss the functions of two distinct domains, radical formation in PQQ, and the presumed existence of bound quinone in mGDH.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Structure at 1.9 A resolution of a quinohemoprotein alcohol dehydrogenase from Pseudomonas putida HK5

The type II quinohemoprotein alcohol dehydrogenase of Pseudomonas putida is a periplasmic enzyme that oxidizes substrate alcohols to the aldehyde and transfers electrons first to pyrroloquinoline quinone (PQQ) and then to an internal heme group. The 1.9 A resolution crystal structure reveals that the enzyme contains a large N-terminal eight-stranded beta propeller domain (approximately 60 kDa) similar to methanol dehydrogenase and a small C-terminal c-type cytochrome domain (approximately 10 kDa) similar to the cytochrome subunit of p-cresol methylhydoxylase. The PQQ is bound near the axis of the propeller domain about 14 A from the heme. A molecule of acetone, the product of the oxidation of isopropanol present during crystallization, appears to be bound in the active site cavity.     

Dopamine beta-hydroxylase from bovine adrenal medulla contains covalently-bound pyrroloquinoline quinone

Treatment of homogeneous dopamine beta-hydroxylase (DBH) preparations from bovine adrenals with the inhibitor phenylhydrazine (PH) changed the structureless absorption spectrum of DBH into spectra with a maximum at 350 nm. A product with this absorption spectrum could be detached with pronase, enabling its isolation. It appeared to be the C(5) hydrazone of pyrroloquinoline quinone (PQQ) and PH, as judged from its properties and the fact that it could be transformed into PQQ itself. From the yield obtained a ratio of 0.85 PQQ per enzyme subunit was calculated. In contrast to copper-quinoprotein amine oxidases (EC 1.4.3.6), hydrazone formation in DBH did not require saturation of the mixture with O2. DBH is the first copper-quinoprotein hydroxylase found so far. The implications of this finding for the current views on mechanism of action and inhibition by hydrazines are discussed. The success of the recently developed 'hydrazine method' [(1987) FEBS Lett. 221, 299-304] for all different types of amine oxidoreductases, suggest that the method could also be applied to other enzymes for which hydrazines are inhibitors and where the identity of the cofactors has not been established or the presence of PQQ is suspected.     

Involvement of a quinoprotein (PQQ-containing) alcohol dehydrogenase in the degradation of polypropylene glycols by the bacterium Stenotrophomonas maltophilia

Previous work has shown that when the bacterium Stenotrophomonas maltophilia is grown on polypropylene glycol, different dye-linked polypropylene glycol dehydrogenase (PPG-DH) activities are induced during growth. Here the purification and characterization of the dehydrogenase activity induced in the stationary phase, and present in the periplasmic space, is described. The homogeneous enzyme preparation obtained consists of a homodimeric protein with a molecular mass of about 123 kDa and an isoelectric point of 5.9. The cofactor of the enzyme appeared to be pyrroloquinoline quinone (PQQ), no heme c was present, and holo-enzyme contained two PQQ molecules per enzyme molecule. In these respects, PPG-DH described here is similar to already known quinoprotein alcohol dehydrogenases, but in other respects, it is different. Therefore, it is suggested that PPG-DH could be a new type of quinoprotein alcohol dehydrogenase. Based on its strong preference for polyols, PPG-DH seems well fitted to carry out the first step in the degradation of PPGs, synthetic polymers containing a variety of hydroxyl groups.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Quinoproteins: structure, function, and biotechnological applications

A new class of oxidoreductase containing an amino acid-derived o-quinone cofactor, of which the most typical is pyrroloquinoline quinone (PQQ), is called quinoproteins, and has been recognized as the third redox enzyme following pyridine nucleotide- and flavin-dependent dehydrogenases. Some quinoproteins include a heme c moiety in addition to the quinone cofactor in the molecule and are called quinohemoproteins. PQQ-containing quinoproteins and quinohemoproteins have a common structural basis, in which PQQ is deeply embedded in the center of the unique superbarrel structure. Increased evidence for the structure and function of quinoproteins has revealed their unique position within the redox enzymes with respect to catalytic and electron transfer properties, and also to physiological and energetic function. The peculiarities of the quinoproteins, together with their unique substrate specificity, have encouraged their biotechnological application in the fields of biosensing and bioconversion of useful compounds, and also to environmental treatment.     

Pyrroloquinoline quinone, a method for its isolation and identification by mass spectrometry.

Procedures for the unambiguous detection and for the isolation and mass spectrometric identification of pyrroloquinoline quinone (PQQ) are presented. The procedure involved acid hydrolysis of protein in the presence of phenylhydrazine and successive isolation and identification of the formed adduct using mass spectrometry. In HPLC the phenylhydrazone of PQQ gave many methylated products, of which the predominant compound was the pentamethylated derivative. After reaction of the phenylhydrazone derivative of PQQ (PHPQQ) with ammonia, a product was obtained which did not contain phenylhydrazine and which formed a pentamethylated derivative as the main methylation product. The HPLC profiles of the methylated products of PHPQQ and of its ammonia derivative were very characteristic and could be used for identification in addition to mass spectrometry. However, prolonged treatment of proteins with phenylhydrazine during hydrolysis can result in the formation of a material that resembles PQQ in some aspects of its behaviour. Thus, analysis by MS is essential for unambiguous identification. This analytical procedure was applied to pig plasma benzylamine oxidase, pig aorta lysyl oxidase, pig kidney diamine oxidase and bovine serum albumin with negative results. However, samples of pronase contained variable quantities of non-covalently bound PQQ: this can lead to erroneous identification of PQQ in enzyme after pronase digestion.     

Glu742 substitution to Lys enhances the EDTA tolerance ofEscherichia coli PQQ glucose dehydrogenase

Summary Based on homology analysis of the PQQ (pyrroloquinoline quinone) glucose dehydrogenase (PQQGDH) gene fromEscherichia coli andAcinetobacter calcoaceticus, Glu742 was substituted to Lys by site directed mutagenesis of theE. coli PQQGDH gene (gcd). The mutant enzyme, E742K showed higher tolerance towards EDTA inactivation than wild type PQQGDH. This is the first mutagenesis study of putative a PQQ binding site in PQQ enzyme.     

Adduct formation of pyrroloquinoline quinone and amino acid

When pyrroloquinoline quinone (PQQ) is mixed with an amino acid, a corresponding Schiff base PQQ adduct is readily formed between carbonyl groups of PQQ and the primary amino group. A potent growth stimulating effect for microorganisms was observed with the PQQ adduct when it was administered in a culture medium. Although PQQ itself shows a marked growth stimulating effect, PQQ adducts appeared to be more active than authentic PQQ when compared on a molar basis. Conversely, unlike authentic PQQ, PQQ adducts were shown to be less active (greater than or equal to 100-fold) as the prosthetic group for a quinoprotein apo-glucose dehydrogenase when examined by holoenzyme formation by exogenous addition of PQQ or PQQ adducts. These observations suggested that PQQ adduct formation readily occurs during isolation procedures for PQQ from biological materials or PQQ - chromophore from quinoproteins. Therefore, the presence of such adducts gives a PQQ estimation much lower than theoretically expected. As an example, formation, isolation and characterization of PQQ - serine are described.     

Nitrile hydratase is a quinoprotein. A possible new function of pyrroloquinoline quinone: activation of H2O in an enzymatic hydration reaction

Nitrile hydratase has been proved to be a quinoprotein with pyrroloquinoline quinone (PQQ) as a prosthetic group. The broad shoulder from 300 to 500 nm in the absorption spectrum of Brevibacterium nitrile hydratase suggested the presence of PQQ. Since PQQ was attached to the enzyme through a covalent linkage, the chromophores were isolated by acid hydrolysis, protease digestion and successive chromatographic separation. The isolated chromophores showed the similar spectroscopic characteristics to those of obtained from the amine oxidase of Aspergillus niger, in which PQQ is covalently linked. The isolated chromophores potently activated apo-D-glucose dehydrogenase (EC 1.1.99.17), supporting the presence of PQQ or a PQQ-like compound in nitrile hydratase. The finding of PQQ in nitrile hydratase strongly suggests a new function of PQQ, i.e., the activation of H2O in the enzymatic hydration reaction.     

Role of calcium in the reaction between pyrroloquinoline quinone and pyridine nucleotides monomers and dimers.

Redox reactions were carried out in aerobiosis and anaerobiosis between NAD(P) dimers or NAD(P)H and pyrroloquinoline quinone (PQQ) in different buffers. The buffer system and pH significantly affected the oxidation rates of nucleotides and the ESR signal intensity of the PQQ(*) radical formed in anaerobiosis by comproportion between the quinone and quinol forms. The relative reactivity of the four nucleotides toward PQQ was affected by pH and buffer nature. PQQ, which behaves as an electron shuttle from nucleotides to oxygen, was first converted to PQQH(2) and then rapidly reoxidized by oxygen, with formation of hydrogen peroxide. Both NAD(P) dimers and NAD(P)H consumed 1 mol of oxygen per mole of reacted molecule of pyridine nucleotide, yielding 1 or 2 mol of NAD(P)(+) from NAD(P)H or from NAD(P) dimers, respectively. Chelating agents such as EDTA and phytate strongly decreased the reaction rate and the PQQ(*) radical signal intensity. Kinetics carried out in the presence of metal ions showed instead an increased reaction rate in the order Ca(2+) > Mg(2+) > Na(+) > K(+). Spectrofluorimetric measurements of PQQ with increasing concentrations of Ca(2+) showed a fluorescence quenching and shift of the maximum emission toward lower wavelengths, while other metal ions showed minor effects, if any. Therefore, it is demonstrated that Ca(2+) binds to PQQ, probably forming a complex which is more reactive with both one-electron (NAD(P) dimers) or two-electron donors (NAD(P)H) in nonenzymic reactions. It is important to recall that Ca(2+) was already found to play active role in PQQ-containing enzymes.     

On the biosynthesis of free and covalently bound PQQ. Glutamic acid decarboxylase from Escherichia coli is a pyridoxo-quinoprotein

Analysis of glutamic acid decarboxylase (GDC) (EC 4.1.1.15) from Escherichia coli ATCC 11246 revealed the presence of six pyridoxal phosphates (PLPs) as well as six covalently bound pyrroloquinoline quinones (PQQs) per hexameric enzyme molecule. This is the second example of a pyridoxo-quinoprotein, suggesting that other atypical pyridoxoproteins (PLP-containing enzymes) have similar cofactor composition. Since the organism did not produce free PQQ and its quinoprotein glucose dehydrogenase was present in the apo form, free PQQ is not used in the assemblage of GDC. Most probably, biosynthesis of covalently bound cofactor occurs in situ via a route which is different from that of free PQQ. Thus, organisms previously believed to be unable to synthesize (free) PQQ could in fact be able to produce quinoproteins with covalently bound cofactor. Implications for the role of PQQ in eukaryotic cells are discussed.     

Pyrroloquinoline quinone (PQQ) is reduced to pyrroloquinoline quinol (PQQH2) by vitamin C,and PQQH2 produced is recycled to PQQ by air oxidation in buffer solution at pH 7.4

Measurements of the reaction of sodium salt of pyrroloquinoline quinone (PQQNa₂) with vitamin C (Vit C) were performed in phosphate-buffered solution (pH 7.4) at 25 °C under nitrogen atmosphere, using UV–vis spectrophotometry. The absorption spectrum of PQQNa₂ decreased in intensity due to the reaction with Vit C and was changed to that of pyrroloquinoline quinol (PQQH₂, a reduced form of PQQ). One molecule of PQQ was reduced by two molecules of Vit C producing a molecule of PQQH₂ in the buffer solution. PQQH₂, thus produced, was recycled to PQQ due to air oxidation. PQQ and Vit C coexist in many biological systems, such as vegetables, fruits, as well as in human tissues. The results obtained suggest that PQQ is reduced by Vit C and functions as an antioxidant in biological systems, because it has been reported that PQQH₂ shows very high free-radical scavenging and singlet-oxygen quenching activities in buffer solutions.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

Nutritional complementation of oxidative glucose metabolism in Escherichia coli via pyrroloquinoline quinone-dependent glucose dehydrogenase and the Entner-Doudoroff pathway.

Two glucose-negative Escherichia coli mutants (ZSC113 and DF214) were unable to grow on glucose as the sole carbon source unless supplemented with pyrroloquinoline quinone (PQQ). PQQ is the cofactor for the periplasmic enzyme glucose dehydrogenase, which converts glucose to gluconate. Aerobically, E. coli ZSC113 grew on glucose plus PQQ with a generation time of 65 min, a generation time about the same as that for wild-type E. coli in a defined glucose-salts medium. Thus, for E. coli ZSC113 the Enter-Doudoroff pathway was fully able to replace the Embden-Meyerhof-Parnas pathway. In the presence of 5% sodium dodecyl sulfate, PQQ no longer acted as a growth factor. Sodium dodecyl sulfate inhibited the formation of gluconate from glucose but not gluconate metabolism. Adaptation to PQQ-dependent growth exhibited long lag periods, except under low-phosphate conditions, in which the PhoE porin would be expressed. We suggest that E. coli has maintained the apoenzyme for glucose dehydrogenase and the Entner-Doudoroff pathway as adaptations to an aerobic, low-phosphate, and low-detergent aquatic environment.     

Covalently bound pyrroloquinoline quinone is the organic prosthetic group in human placental lysyl oxidase.

Treatment of purified human placental lysyl oxidase with 2,4-dinitrophenylhydrazine (DNPH) resulted in a large spectral change and inhibition of enzyme activity. Proteolytic degradation of the derivatized enzyme yielded only one single coloured product, which was spectrally and chromatographically identical with the C-5 hydrazone of PQQ (pyrroloquinoline quinone) and DNPH. Since this represents the first example of a PQQ-containing enzyme in man, possible implications of the finding are discussed.     

Pyrroloquinoline quinone, a chemotactic attractant for Escherichia coli.

Escherichia coli is attracted by pyrroloquinoline quinone (PQQ), and chemotaxis toward glucose is enhanced by the presence of PQQ. A ptsI mutant showed no chemotactic response to either glucose or PQQ alone but did show a chemotactic response to a mixture of glucose and PQQ. A strain lacking the methylated chemotaxis receptor protein Tar showed no response to PQQ.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.