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.     

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.     

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.     

Crystal structure of l-sorbose dehydrogenase,a pyrroloquinoline quinone-dependent enzyme with homodimeric assembly,from Ketogulonicigenium vulgare

The crystal structure of the l-sorbose dehydrogenase (SDH) from Ketogulonicigenium vulgare Y25 has been determined at 2.7 Å resolution using the molecular replacement method. The overall structure of SDH is similar to that of other quinoprotein dehydrogenases; consisting of an eight bladed β-propeller PQQ domain and protrusion loops. We identified a stable homodimer in crystal and demonstrated its existence in solution by sedimentation velocity measurement. By biochemical characterization of the SDH in vitro, using l-sorbose as substrate and cytochrome c551 as electron acceptor, we revealed cytochrome c551 acting as physiological primary electron acceptor for SDH.     

Pyrroloquinoline Quinone-Dependent Cytochrome Reduction in Polyvinyl Alcohol-Degrading Pseudomonas sp. Strain VM15C

A polyvinyl alcohol (PVA) oxidase-deficient mutant of Pseudomonas sp. strain VM15C, strain ND1, was shown to possess PVA dehydrogenase, in which pyrroloquinoline quinone (PQQ) functions as a coenzyme. The mutant grew on PVA and required PQQ for utilization of PVA as an essential growth factor. Incubation of the membrane fraction of the mutant with PVA caused cytochrome reduction of the fraction. Furthermore, it was found that in spite of the presence of PVA oxidase, the membrane fraction of strain VM15C grown on glucose without PQQ required PQQ for cytochrome reduction during incubation with PVA. The results provide evidence that PVA dehydrogenase couples with the electron transport chain of PVA-degrading bacteria but that PVA oxidase does not.     

Localization of Polyvinyl Alcohol Oxidase Produced by a Bacterial Symbiont, Pseudomonas sp. Strain VM15C

An axenic culture of a polyvinyl alcohol (PVA)-degrading symbiont, Pseudomonas sp. strain VM15C, was established on PVA with a crude preparation of the growth factor (factor A) produced by the symbiotic partner Pseudomonas putida VM15A. An increase of factor A in the culture medium enhanced the cell-associated PVA oxidase activity as well as the growth rate, but decreased production of extracellular PVA oxidase. PVA oxidase in cells grown on PVA was present in the periplasmic space at a higher ratio than in cells grown on peptone. PVA degradation occurred rapidly with washed cells. PVA was also degraded by immobilized cells entrapped in agar gels.     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.     

Enhancement of Pyrroloquinoline Quinone Production and Polyvinyl Alcohol Degradation in Mixed Continuous Cultures of Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C with Mixed Carbon Sources

In a mixed continuous culture of Pseudomonas putida VM15A and Pseudomonas sp. strain VM15C with polyvinyl alcohol (PVA) as the sole source of carbon, growth of the PVA-degrading bacterium VM15C and, hence, PVA degradation were limited by the growth factor, pyrroloquinoline quinone, produced by VM15A. Feeding of a carbon source for VM15A, ethanol, with PVA enhanced pyrroloquinoline quinone production and caused increases in the VM15C population and PVA degradation in a mixed continuous culture. There was an optimum range for PVA degradation of the ethanol concentration, although pyrroloquinoline quinone concentrations in continuous mixed cultures increased with increasing ethanol concentration.     

A novel caffeine dehydrogenase in Pseudomonas sp. strain CBB1 oxidizes caffeine to trimethyluric acid

A unique heterotrimeric caffeine dehydrogenase was purified from Pseudomonas sp. strain CBB1. This enzyme oxidized caffeine to trimethyluric acid stoichiometrically and hydrolytically, without producing hydrogen peroxide. The enzyme was not NAD(P)⁺ dependent; coenzyme Q₀ was the preferred electron acceptor. The enzyme was specific for caffeine and theobromine and showed no activity with xanthine.     

Purification and properties of a polyvinyl alcohol-degrading enzyme produced by a strain of Pseudomonas

An enzyme which degraded polyvinyl alcohol, a water-soluble synthetic polymer, was isolated as a single protein from a culture of a strain of Pseudomonas. The pink-colored enzyme had absorption maxima at 280, 370, and 480 nm, a molecular weight of about 30,000, and an isoelectric point at about pH 10.3. The enzyme was most active at pH values from 7 to 9 and at 40 dgC and was stable at pH values from 3.5 to 9.5 and at temperatures below 45 dgC. The viscosity of the reaction mixture decreased and the pH dropped when the enzyme acted on polyvinyl alcohol as a substrate. Furthermore, the enzyme required O₂ for the reaction and produced 1 mol of H₂O₂, per 1 mol of O₂ consumed. The molecules of polyvinyl alcohol were cleaved into small fragments with a wide distribution of molecular weights. Inorganic Hg ions markedly inactivated the enzyme, and the activity was immediately recovered by glutathione. Enzyme inhibitors tested, which included p-chloromercuribenzoic acid, KCN, o-phenanthroline, and H₂O₂, showed no effect on the activity. Polyvinyl alcohol oxidized by periodic acid was similarly oxidized by the enzyme. The enzyme did not oxidize most of a variety of low molecular weight hydroxy compounds examined, such as primary alcohols, secondary alcohols, tertiary alcohols, diols, triols, and polyols, except for some secondary alcohols, such as 4-heptanol.     

The atzB gene of Pseudomonas sp. strain ADP encodes the second enzyme of a novel atrazine degradation pathway.

We previously reported the isolation of a 21.5-kb genomic DNA fragment from Pseudomonas sp. strain ADP, which contains the atzA gene, encoding the first metabolic step for the degradation of the herbicide atrazine (M. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, we show that this fragment also contained the second gene of the atrazine metabolic pathway, atzB. AtzB catalyzed the transformation of hydroxyatrazine to N-isopropylammelide. The product was identified by use of high-performance liquid chromatography, mass spectrometery, and nuclear magnetic resonance spectroscopy. Tn5 mutagenesis of pMD1 was used to determine that atzB was located 8 kb downstream of atzA. Hydroxyatrazine degradation activity was localized to a 4.0-kb ClaI fragment, which was subcloned into the vector pACYC184 to produce plasmid pATZB-2. The DNA sequence of this region was determined and found to contain two large overlapping divergent open reading frames, ORF1 and ORF2. ORF1 was identified as the coding region of atzB by demonstrating that (i) only ORF1 was transcribed in Pseudomonas sp. strain ADP, (ii) a Tn5 insertion in ORF2 did not disrupt function, and (iii) codon usage was consistent with ORF1 being translated. AtzB had 25% amino acid identity with TrzA, a protein that catalyzes a hydrolytic deamination of the s-triazine substrate melamine. The atzA and atzB genes catalyze the first two steps of the metabolic pathway in a bacterium that rapidly metabolizes atrazine to carbon dioxide, ammonia, and chloride.     

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.     

FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme from Mycobacterium sp. strain Takeo. Purification and some properties

FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme, was homogeneously purified from the particulate fraction of Mycobacterium sp. strain Takeo. The isolated enzyme contains no FAD and few phospholipid, and has a specific activity of 300-360 units/mg of protein. In the assay system without addition of phospholipid (cardiolipin), the enzyme activity was only about 3% of maximum activity. The molecular weight was estimated to be 51 000-55 000 by four methods. Titration by p-chloromercuribenzoate revealed the presence of one cysteine residue/mol of enzyme. The isoelectric point was found to be pH 6.9 by isoelectric focusing. From circular dichroism spectral data, the enzyme protein was found to contain alpha-helix structure of 24%.     

Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816.

The initial reactions in the oxidation of naphthalene by Pseudomonas sp. strain NCIB 9816 involves the enzymatic incorporation of one molecule of oxygen into the aromatic nucleus to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme catalyzing this reaction, naphthalene dioxygenase, was resolved into three protein components, designated A, B, and C, by DEAE-cellulose chromatography. Incubation of naphthalene with components A, B, and C in the presence of NADH resulted in the formation of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The ratio of oxygen and NADH utilization to product formation was 1:1:1. NADPH also served as an electron donor for naphthalene oxygenation. However, its activity was less than 50% of that observed with NADH. Component A showed NAD(P)H-cytochrome c reductase activity which was stimulated by the addition of flavin adenine dinucleotide and flavin mononucleotide. A similar stimulation was observed when these flavin nucleotides were added to the naphthalene dioxygenase assay system. These preliminary observations indicate that naphthalene dioxygenase has properties in common with both monooxygenase and dioxygenase multicomponent enzyme systems.     

Oxidation of biphenyl by a multicomponent enzyme system from Pseudomonas sp. strain LB400.

Pseudomonas sp. strain LB400 grows on biphenyl as the sole carbon and energy source. This organism also cooxidizes several chlorinated biphenyl congeners. Biphenyl dioxygenase activity in cell extract required addition of NAD(P)H as an electron donor for the conversion of biphenyl to cis-2,3-dihydroxy-2,3-dihydrobiphenyl. Incorporation of both atoms of molecular oxygen into the substrate was shown with 18O2. The nonlinear relationship between enzyme activity and protein concentration suggested that the enzyme is composed of multiple protein components. Ion-exchange chromatography of the cell extract gave three protein fractions that were required together to restore enzymatic activity. Similarities with other multicomponent aromatic hydrocarbon dioxygenases indicated that biphenyl dioxygenase may consist of a flavoprotein and iron-sulfur proteins that constitute a short electron transport chain involved in catalyzing the incorporation of both atoms of molecular oxygen into the aromatic ring.     

Characterization of partially purified alcohol dehydrogenase from Rhodococcus sp. strain GK1

An NAD-dependent secondary alcohol dehydrogenase (ADH) produced by Rhodococcus sp. GK1 was purified about fivefold with a yield of 82% by hydrophobic interaction chromatography. This enzyme reduced monoketones, diketones and α-dicarbonyl compounds ; it oxidized secondary alcohols but not primary alcohols. Optimum pH was 7·0 or 8·5 for reduction or oxidation of substrates, respectively, and optimal temperature for activity was 55 °C. The apparent molecular mass of ADH was about 60 kDa by gel filtration chromatography.     

Kinetics study of pyridine biodegradation by a novel bacterial strain,Rhizobium sp. NJUST18

Biodegradation of pyridine by a novel bacterial strain, Rhizobium sp. NJUST18, was studied in batch experiments over a wide concentration range (from 100 to 1,000 mg l^?1). Pyridine inhibited both growth of Rhizobium sp. NJUST18 and biodegradation of pyridine. The Haldane model could be fitted to the growth kinetics data well with the kinetic constants μ* = 0.1473 h^?1, K _s = 793.97 mg l^?1, K _i = 268.60 mg l^?1 and S _m = 461.80 mg l^?1. The true μ _max, calculated from μ*, was found to be 0.0332 h^?1. Yield coefficient Y _X/S depended on S _i and reached a maximum of 0.51 g g^?1 at S _i of 600 mg l^?1. V _max was calculated by fitting the pyridine consumption data with the Gompertz model. V _max increased with initial pyridine concentration up to 14.809 mg l^?1 h^?1. The q _S values, calculated from $V_{ \hbox{max} }$ , were fitted with the Haldane equation, yielding q _Smax = 0.1212 g g^?1 h^?1 and q* = 0.3874 g g^?1 h^?1 at S _m′ = 507.83 mg l^?1, K _s′ = 558.03 mg l^?1, and K _i′ = 462.15 mg l^?1. Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, μ _max and S _m obtained for Rhizobium sp. NJUST18 were relatively high. High K _i and K _i′ values and extremely high K _s and K _s′ values indicated that NJUST18 was able to grow on pyridine within a wide concentration range, especially at relatively high concentrations.