Pig kidney dopa decarboxylase. Structure and function.
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Amino Acid Sequence↗
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PQQ and electron transport in mammalian systems.
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Animals↗
Is PQQ a significant nutrient in addition to its role as a therapeutic agent in the higher animal?
The controversy as to the nutritional status of pyroloquinoline (PQQ) has now been broadened to include various animal tissues by evidence of its vitamin status in mice, a mammalian PQQ reductase, and evidence that this enzyme participates in mechanisms protecting tissues against oxidative stress.
Animals↗
The role of phenolics, conjugated linoleic acid, carnosine, and pyrroloquinoline quinone as nonessential dietary antioxidants.
Oxidative reactions have been implicated in the development of numerous diseases including atherosclerosis and cancer. Oxidation of lipids, proteins, and nucleic acids can result in loss of membrane integrity and function, inactivation of enzymes, modification of lipoproteins, and chemical alteration of DNA. Active oxygen species, transition metals, reducing agents, and enzymes such as lipoxygenase are all involved in the catalysis of oxidative reactions. Since lipid oxidation catalysts and active oxygen species are ubiquitous to all biological systems and since lipid oxidation products can enter the body via oxidized foods, numerous endogenous antioxidant systems have been developed. Endogenous antioxidant systems include antioxidant enzymes, free radical scavengers, and metal chelators. The purpose of this review is to examine the potential of nonessential dietary components that inhibit oxidative reactions in foods and biological tissues.
Animals↗
Nutritional importance of pyrroloquinoline quinone.
Mice fed a chemically defined diet devoid of pyrroloquinoline quinone (PQQ) grew poorly, failed to reproduce, and became osteolathyritic. Moreover, severely affected mice had friable skin, skin collagen that was readily extractable into neutral salt solutions, and decreased lysyl oxidase. The identification of functional defects in connective tissue and the growth retardation associated with PQQ deprivation suggest that PQQ plays a fundamental role as a growth factor or vitamin.
Animals↗
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.
Alcohol Oxidoreductases↗
Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.
Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.
Amino Acid Sequence↗
Degradation of tetrahydrofurfuryl alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-dependent alcohol dehydrogenase.
An organism tentatively identified as Ralstonia eutropha was isolated from enrichment cultures containing tetrahydrofurfuryl alcohol (THFA) as the sole source of carbon and energy. The strain was able to tolerate up to 200 mM THFA in mineral salt medium. The degradation was initiated by an inducible ferricyanide-dependent alcohol dehydrogenase (ADH) which was detected in the soluble fraction of cell extracts. The enzyme catalyzed the oxidation of THFA to the corresponding tetrahydrofuran-2-carboxylic acid. Studies with n-pentanol as the substrate revealed that the corresponding aldehyde was released as a free intermediate. The enzyme was purified 211-fold to apparent homogeneity and could be identified as a quinohemoprotein containing one pyrroloquinoline quinone and one covalently bound heme c per monomer. It was a monomer of 73 kDa and had an isoelectric point of 9.1. A broad substrate spectrum was obtained for the enzyme, which converted different primary alcohols, starting from C2 compounds, secondary alcohols, diols, polyethylene glycol 6000, and aldehydes, including formaldehyde. A sequence identity of 65% with a quinohemoprotein ADH from Comamonas testosteroni was found by comparing 36 N-terminal amino acids. The ferricyanide-dependent ADH activity was induced during growth on different alcohols except ethanol. In addition to this activity, an NAD-dependent ADH was present depending on the alcohol used as the carbon source.
Alcaligenes↗
Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid.
A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.
Aldehyde Oxidoreductases↗
Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).
The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.
Acinetobacter↗
Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.
D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.
Electron Transport Complex IV↗
Cloning of the genes involved in synthesis of coenzyme pyrrolo-quinoline-quinone from Acinetobacter calcoaceticus.
Mutants of Acinetobacter calcoaceticus LMD79.41 were isolated that are defective in the synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ). A gene bank of the wild-type. A. calcoaceticus genome was constructed with the binary plasmid system pLV21-RP4 delta Km. The DNA of A. calcoaceticus LMD79.41 was partially digested with Sau3A, and fragments of about 15 kilobases were inserted into the BamHI site of pLV21. The hybrid plasmids maintained in Escherichia coli were transferred by conjugation to the PQQ- mutants of A. calcoaceticus. One hybrid plasmid was isolated that complements all isolated PQQ- mutants. Subcloning of this plasmid in the vector pRK290 resulted in an insert of 5 kilobases on which at least four different genes involved in PQQ synthesis could be indicated. With Tn5 insertions the four PQQ genes were mapped, and it was shown that these genes are most probably located in three operons.
Acinetobacter↗
Acinetobacter calcoaceticus genes involved in biosynthesis of the coenzyme pyrrolo-quinoline-quinone: nucleotide sequence and expression in Escherichia coli K-12.
Synthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ) from Acinetobacter calcoaceticus requires the products of at least four different genes. In this paper we present the nucleotide sequence of a 5,085-base-pair DNA fragment containing these four genes. Within the DNA fragment three reading frames are present, coding for proteins of Mr 10,800, 29,700, and 43,600 and corresponding to three of the PQQ genes. In the DNA region where the fourth PQQ gene was mapped the largest possible reading frame encodes for a polypeptide of only 24 amino acids. Still, the expression of this region is essential for the biosynthesis of PQQ. A possible role for this DNA region is discussed. Sandwiched between two PQQ genes an additional reading frame is present, coding for a protein of Mr 33,600. This gene, which is probably transcribed in the same operon as three of the PQQ genes, seems not required for PQQ synthesis. Expression of the PQQ genes in Acinetobacter lwoffi and Escherichia coli K-12 led to the synthesis of the coenzyme in these organisms.
Acinetobacter↗
A 24-amino-acid polypeptide is essential for the biosynthesis of the coenzyme pyrrolo-quinoline-quinone.
At least four genes are required for the biosynthesis of the coenzyme pyrrolo-quinoline-quinone (PQQ) in Acinetobacter calcoaceticus. The DNA region where one of these genes was mapped codes for a polypeptide of only 24 amino acids. Here we show that indeed this small peptide is essential for PQQ synthesis. Site-directed mutagenesis shows that at least one glutamate and one tyrosine residue of the polypeptide are essential for its function.
Acinetobacter calcoaceticus↗
Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A.
The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.
Acinetobacter↗
Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC.
Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the presence of PQQ. These putative PQQ biosynthesis mutants have been complemented by using previously isolated clones containing M. extorquens AM1 DNA, which were known to contain genes necessary for oxidation of methanol to formaldehyde (mox genes). Subcloning and transposon mutagenesis experiments have assigned these mutants to five complementation groups in two gene clusters. Representatives of each complementation group were shown to lack detectable PQQ in the growth medium and in cell extracts and to contain methanol dehydrogenase polypeptides that were inactive. Therefore, these mutants all appear to be defective in PQQ biosynthesis. PQQ biosynthesis mutants of Methylobacterium organophilum DSM 760 and M. organophilum XX were complemented by using M. extorquens AM1 subclones, and PQQ biosynthesis mutants of M. extorquens AM1 and M. organophilum XX were complemented by using M. organophilum DSM 760 subclones. This analysis suggested that a total of six PQQ biosynthesis complementation groups were present in M. extorquens AM1 and M. organophilum DSM 760. A 2-kb M. extorquens AM1 DNA fragment that complemented the MoxO class of PQQ biosynthesis mutants was sequenced and found to contain two complete open reading frames and the N-terminal sequence of a third. These genes designated pqqDGC, had predicted gene products with substantial similarity to the gene products of corresponding pqq genes in Acinetobacter calcoaceticus and Klebsiella pneumoniae. pqqD encodes a 29-amino-acid peptide which contains a tyrosine residue and glutamate residue that are conserved in the equivalent peptides of K. pneumoniae, PqqA (23 amino acids), and A. calcoaceticus, PqqIV (24 amino acids), and are thought to be the precursors for PQQ biosynthesis. The organizations of a cluster of five PQQ biosynthetic genes appear to be similiar in four different bacteria (M. extorquens AM1, M. organophilum DSM 760, K. pneumoniae, and A. calcoaceticus). Our results show that a total of seven pqq genes are present in M. extorquens AM1, and these have been designated pqqDGCBA and pqqEF.
Amino Acid Sequence↗
Transcriptional analysis of pqqD and study of the regulation of pyrroloquinoline quinone biosynthesis in Methylobacterium extorquens AM1.
Methanol dehydrogenase, the enzyme that oxidizes methanol to formaldehyde in gram-negative methylotrophs, contains the prosthetic group pyrroloquinoline quinone (PQQ). To begin to analyze how the synthesis of PQQ is coordinated with the production of other methanol dehydrogenase components, the transcription of one of the key PQQ synthesis genes has been studied. This gene (pqqD) encodes a 29-amino-acid peptide that is thought to be the precursor for PQQ biosynthesis. A unique transcription start site was mapped to a guanidine nucleotide 95 bp upstream of the pqqD initiator codon. RNA blot analysis identified two transcripts, a major one of 240 bases encoding pqqD and a minor one of 1,300 bases encoding pqqD and the gene immediately downstream, pqqG. Both transcripts are present at similar levels in cells grown on methanol and on succinate, but the levels of PQQ are about fivefold higher in cells grown on methanol than in cells grown on succinate. These results suggest that PQQ production is regulated at a level different from the transcription of pqqD. The genes mxbM, mxbD, mxcQ, mxcE, and mxaB are required for transcription of the genes encoding the methanol dehydrogenase subunits and were assessed for their role in PQQ production. PQQ levels were measured in mutants defective in each of these regulatory genes and compared with levels of pqqD transcription, measured with a transcriptional fusion between the pqqD promoter and xylE. The results showed that only a subset of these regulatory genes (mxbM, mxbD, and mxaB) is required for transcription of pqqD, and only mxbM and mxbD mutants affected the final levels of PQQ significantly.
Alcohol Oxidoreductases↗
Generation mechanism and purification of an inactive form convertible in vivo to the active form of quinoprotein alcohol dehydrogenase in Gluconobacter suboxydans.
Alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound quinohemoprotein-cytochrome c complex involved in vinegar production. In Gluconobacter suboxydans grown under acidic growth conditions, it was found that ADH content in the membranes was largely increased but the activity was not much changed, suggesting that such a condition produces an inactive form of ADH (inactive ADH). A similar phenomenon could be also observed in Acetobacter aceti, another genus of acetic acid bacteria. Furthermore, aeration conditions were also shown to affect ADH production; the ADH level was increased and was present as an active form under low-aeration conditions, while the ADH level was decreased and was present mainly as an inactive form under high-aeration conditions. Inactive ADH was solubilized from the membranes of G. suboxydans grown in acidic and high-aeration conditions and was purified separately from the normal, active form of ADH (active ADH). In spite of having 10 times less enzyme activity than active ADH, inactive ADH could not be distinguished from active ADH with respect to their subunit compositions, molecular sizes, and prosthetic groups. Inactive ADH, however, had a relatively loose conformation with a partially oxidized state, while active ADH had a tight conformation with a completely reduced state, suggesting that inactive ADH may lack a right subunit's interaction and that one of the heme c components may be inactivated. Reactivation from such an inactive ADH occurred either by shifting of the pH of the culture medium up during the cultivation or by incubation of the resting cells at the neutral pH region in the presence of an energy source such as D-sorbitol. Such an activation of ADH was repressed by the addition of a proton uncoupler and could not occur in the spheroplasts. Thus, the results suggest that inactive ADH could be generated abundantly under acidic growth conditions and converted to the active form at a neutral culture pH. The data also suggest that some periplasmic component may be involved in the conversion of inactive ADH into the active form by consuming some forms of energy.
Acetobacteraceae↗