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PubMed2004

Energy-dependent transformation of F0.F1-ATPase in Paracoccus denitrificans plasma membranes.

F(0).F(1)-ATP synthase in tightly coupled inside-out vesicles derived from Paracoccus denitrificans catalyzes rapid respiration-supported ATP synthesis, whereas their ATPase activity is very low. In the present study, the conditions required to reveal the Deltamu(H+)-generating ATP hydrolase activity of the bacterial enzyme have been elucidated. Energization of the membranes by respiration results in strong activation of the venturicidin-sensitive ATP hydrolysis, which is coupled with generation of Deltamũ(H+). Partial uncoupling stimulates the proton-translocating ATP hydrolysis, whereas complete uncoupling results in inhibition of the ATPase activity. The presence of inorganic phosphate is indispensable for the steady-state turnover of the Deltamũ(H+)-activated ATPase. The collapse of Deltamũ(H+) brings about rapid deactivation of the enzyme, which has been subjected to pre-energization. The rate and extent of the deactivation depend on protein concentration, i.e. the more vesicles are present in the assay mixture, the higher the rate and extent of the deactivation is seen. Sulfite and the ADP-trapping system protect ATPase against the Deltamũ(H+) collapse-induced deactivation, whereas phosphate delays the rate of deactivation. A low concentration of ADP (<1 microm) increases the rate of deactivation. Taken together, the results suggest that latent proton-translocating ATPase in P. denitrificans is kinetically equivalent to the previously characterized ADP(Mg2+)-inhibited, azide-trapped bovine heart mitochondrial F(0).F(1)-ATPase (Galkin, M. A., and Vinogradov, A. D. (1999) FEBS Lett. 448, 123-126). A Deltamũ(H+)-sensitive mechanism operates in P. denitrificans that prevents physiologically wasteful consumption of ATP by F(0).F(1)-ATPase (synthase) complex when the latter is unable to maintain certain value of Deltamũ(H+).

Adenosine Diphosphate↗

PubMed2005

Y25S variant of Paracoccus pantotrophus cytochrome cd1 provides insight into anion binding by d1 heme and a rare example of a critical difference between solution and crystal structures.

Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.

Anions↗

PubMed1996

The isolation and some properties of the membrane-bound lactate dehydrogenase of Paracoccus denitrificans.

Membrane-bound lactate dehydrogenase was isolated from the cells of Paracoccus denitrificans by a combination of phase separation and chromatographic methods. It was NAD(+)-independent; phenazine methosulphate and 2,6-dichlorophenolindophenol could act as electron acceptors. It preferred D-lactate over the L-form (K(m) = 0.81 and 4.40 mM, respectively). Relative molecular weight estimations were 54000 +/- 3000 (electrophoresis) and 50000 +/- 5000 (gel chromatography) It was inhibited by thenoyltrifluoroacetone and diethyl pyrocarbonate, SH-reagents were without effect. No other lactate dehydrogenase activity was found in any subcellular fraction of cells grown on D,L-lactate, L-lactate, glucose or succinate.

Acetone↗

PubMed1997

Iron as a possible mediator of the oxic-to-anoxic transition in Paracoccus denitrificans.

Expression of the lacZ gene from the Fnr-regulated FF-melR promoter on a plasmid in iron-deprived Paracoccus denitrificans cells required not only a decreased oxygen tension but also supplementation with iron. The levels of beta-galactosidase and 5-aminolevulinate synthase showed comparable responses to changes in iron availability. The presence of soluble and particulate enzymes catalyzing the reduction of Fe(III) by NADH suggests a hypothesis in which the redox state of the cytoplasmic NAD-couple determines the concentration of free Fe(II) and thereby modulates the activity of a common regulator of the Fnr type.

5-Aminolevulinate Synthetase↗

PubMed2004

Purification and some properties of isocitrate dehydrogenase from Paracoccus denitrificans.

NADP-dependent isocitrate dehydrogenase (ICDH) from the bacterium Paracoccus denitrificans was purified to homogeneity. The purification procedure involved ammonium sulphate fractionation, ion exchange chromatography, and gel permeation chromatography. The specific activity of purified ICDH was 801 nkat/mg, the yield of the enzyme 58%. The purity of the enzyme was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. ICDH is a dimer composed of two probably identical subunits of relative molecular weight 90,000. The pH optimum of the enzyme reaction in the direction of substrate oxidation was found to be 5.6; the presence of Mn2+ is essential for enzyme activity. The absorption and fluorescence spectra of the homogeneous enzyme were measured as well.

Ammonium Sulfate↗

PubMed1982

The 5S ribosomal RNAs of Paracoccus denitrificans and Prochloron.

The nucleotide sequences of the 5S rRNAs of Paracoccus denitrificans and Prochloron sp. are (formula: see text), respectively. Specific phylogenetic relationships of P. denitrificans with purple non-sulphur bacteria, and of Prochloron with cyanobacteria are demonstrated, and unique features of potential secondary structure are described.

Base Sequence↗

PubMed1997

Paracoccus denitrificans aromatic amino acid aminotransferase: a model enzyme for the study of dual substrate recognition mechanism.

The gene for aromatic amino acid aminotransferase (ArAT) from Paracoccus denitrificans was cloned, sequenced, and overexpressed in Escherichia coli cells. The sequence differed from that reported previously [Takagi, T., Taniguchi, T., Yamamoto, Y., and Shibatani, T. (1991) Biotechnol. Appl. Biochem. 13, 112-119]. The enzyme (pdArAT) was purified to homogeneity, and characterized. It was similar to aspartate aminotransferase (AspAT) and ArAT of E. coli (ecArAT) in many respects, including gross protein structure and spectroscopic properties. pdArAT showed activities toward both dicarboxylic and aromatic substrates, and analysis of the binding of substrate analogs and quasisubstrates to the enzyme showed that both dicarboxylic and aromatic substrates take a similar orientation in the active site of pdArAT; these properties are essentially identical with those of ecArAT. As in the case of ecArAT, neutral amino acids with larger side chains are better substrates for pdArAT, suggesting that hydrophobic interaction between the substrate and the enzyme is important for the recognition of substrates with neutral side chains. pdArAT catalyzed transamination of phenylalanine and tyrosine far more efficiently (10(2)-fold in terms of kcat/Km) than those of straight-chain aliphatic amino acids with similar side-chain surface area, whereas ecArAT did not show significant preference for aromatic amino acids over aliphatic amino acids. This shows that the substrate-side-chain-binding pocket of pdArAT, as compared with the pocket of ecArAT, is well suited in shape for interaction with the phenyl and hydroxyphenyl rings of substrates. Thus, pdArAT is an ideal enzyme among ArATs for the study of the high-specificity recognition of two different kinds of substrates, the one having a carboxylic side chain and the other having an aromatic side chain.

Amino Acid Sequence↗

PubMed1998

Metal chelating properties of adenylate kinase from Paracoccus denitrificans.

Zinc, a common element of adenylate kinases from Gram-positive bacteria, binds to a structural motif consisting of three or four cysteine residues, Cys-X2-Cys-X16-Cys-X2-Cys/Asp. The enzyme from Paracoccus denitrificans, a Gram-negative bacterium, has structural features much similar to those of adenylate kinases from Gram-positive organisms [Spurgin, P., Tomasselli, A.G., and Schiltz, E. (1989) Eur. J. Biochem., 179, 621-628]. However, adenylate kinase isolated from this bacterium was not reported to bind metal. These findings prompted us to clone the corresponding gene of P. denitrificans, and to characterize the enzyme overproduced in Escherichia coli. The deduced primary structure of adenylate kinase from P. denitrificans revealed two differences from that previously published: Cys was found at position 130 instead of His, and His was found at position 138 instead of Gly. The recombinant enzyme is a dimer which binds either zinc or iron, in a metal/monomer ratio of one. The dissociating sulfhydryl reagent, p-(hydroxy-mercuri)phenylsulfonate, released the metal from the protein, confirming that thiols are involved in zinc- or iron-binding. The iron-chelated form of recombinant P. denitrificans adenylate kinase, which is essentially under reduced form, transfers electrons to the oxidized cytochrome c. In conclusion, the absence of metal in the enzyme isolated from P. denitrificans is not related to the protein structure but most probably due to the physiological properties of the host organism.

Adenylate Kinase↗

PubMed1987

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. I. The flux of carbon in energy metabolism and the operation of the TCA cycle.

The metabolism of Paracoccus denitrificans, grown on either [2,3-13C]succinate or [1,4-13C]succinate, was investigated by using gas chromatography-mass spectrometry. The distribution of label in a group of metabolites closely related to the TCA-cycle intermediates showed that the flux of carbon from succinate in energy metabolism in vivo was via pyruvate (malic enzyme) and acetyl CoA. The labelling pattern of the carboxyl groups showed that one fifth of the succinate pool was formed by the regeneration of succinate via the TCA cycle, and four fifths was supplied externally as substrate from the medium.

Carbon Isotopes↗

PubMed1987

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. II. Isoleucine biosynthesis.

Paracoccus denitrificans was grown on either [2,3-13C]succinate or [1,4-13C]succinate, and extracts were analysed by using gas chromatography-mass spectrometry. The distribution of label in isoleucine indicated that the 2-ketobutyrate required for isoleucine biosynthesis was mainly produced from pyruvate by 2-keto-acid chain elongation (i.e. the 'pyruvate elongation pathway'). Approximately 10% of isoleucine was produced by a second pathway involving propionyl CoA. Threonine and glutamate were not utilized by P. denitrificans as a source of 2-ketobutyrate production for isoleucine biosynthesis under the growth conditions used.

Carbon Isotopes↗

PubMed1988

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. III. Biosynthetic pathways.

The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2,3-13C]succinate or [1,4-13C]succinate was investigated by using gas chromatography-mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo. The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C4, C5 and C6 sugars without the use of fructose-1,6-bisphosphate aldolase (which has not been detected in P. denitrificans) is proposed. The 13C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo.

Amino Acids↗

PubMed1993

Transfer of Thiosphaera pantotropha to Paracoccus denitrificans.

Comparative sequence analysis of in vitro-amplified 16S rRNA genes of Thiosphaera pantotropha GB17T (T = type strain) and Paracoccus denitrificans LMG 4218T revealed identical 16S rRNA primary structures for the two organisms. The level of overall DNA similarity of Thiosphaera pantotropha GB17T and P. denitrificans DSM 65T is 85%, as determined by quantitative DNA-DNA hybridization. Therefore, we propose the transfer of Thiosphaera pantotropha to P. denitrificans. The closest relative of Thiosphaera pantotropha and P. denitrificans is Thiobacillus versutus, as revealed by comparative 16S rRNA sequence analysis. These organisms are members of the alpha subclass of the Proteobacteria. Within this subclass, Thiosphaera pantotropha, P. denitrificans, and Thiobacillus versutus form a phylogenetic group with Rhodobacter sphaeroides, Rhodobacter capsulatus, and "Erythrobacter longus."

Bacteria↗

PubMed1985

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans.

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.

Alcohol Dehydrogenase↗

PubMed1989

The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa.

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.

Nitrates↗

PubMed1991

The periplasmic modifier protein for methanol dehydrogenase in the methylotrophs Methylophilus methylotrophus and Paracoccus denitrificans.

A modifier protein (M-protein), which increases the affinity of methanol dehydrogenase (MDH) for alcohols but decreases its affinity for formaldehyde, has been partially purified from Methylophilus methylotrophus and Paracoccus denitrificans. Analysis was complicated by non-protein factors in bacterial extracts that are able to mimic M-protein in one of its functions-that of increasing the activity of MDH with butane-1,3-diol in the dye-linked assay system. The 67 kDa polypeptide, previously identified as a subunit of the M-protein, is an unrelated cytoplasmic protein. The M-protein is exclusively periplasmic and is a multimeric protein with subunits of 45 kDa. The M-protein is active in the 'physiological' assay system with the specific cytochrome c electron acceptor for MDH, lowering its affinity for formaldehyde. It has its maximum effect when the ratio of M-protein:MDH is 1:5 but its concentration in the periplasm is much lower than 20% of that of MDH.

Alcohol Oxidoreductases↗

PubMed1996

Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis.

The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.

Base Sequence↗

PubMed1997

The Paracoccus denitrificans ccmA, B and C genes: cloning and sequencing, and analysis of the potential of their products to form a haem or apo- c-type cytochrome transporter.

Two c-type cytochrome deficient mutants of Paracoccus denitrificans, HN49 and HN53, were isolated by Tn5 mutagenesis and screening for failure to oxidize dimethylphenylenediamine (the Nadi test). Both were completely deficient in c-type cytochromes. Genomic DNA flanking the site of Tn5 insertion in HN53 was cloned by marked rescue and a 3.1 kb region sequenced. Three of the genes, designated ccmA, ccmB and ccmC, present in this region are proposed to encode the components of a membrane transporter of the ABC-(ATP-binding cassette) superfamily, which is similar to a group of transporters postulated to translocate either haem or apocytochromes c. The Tn5 elements in HN49 and HN53 shown to be inserted in ccmB and ccmA, respectively. Sequence analysis suggested that both CcmB and CcmC have the potential to interact with CcmA and thus that the three gene products probably associate to form a complex with (CcmA)2-CcmB-CcmC stoichiometry; it is also indicated a lack of similarity between CcmB and CcmC and the membrane-integral components of transporters mediating uptake of haem or other iron complexes. Supplementation of growth media with haem did not stimulate c-type cytochrome formation in HN49 or HN53, although it elevated levels of soluble haemoproteins and membrane-bound cytochromes b, suggesting that exogenous haem can traverse both outer and inner membranes of P. denitrificans. HN49 and HN53 accumulated apocytochrome C550 to much lower levels than other c-type cytochrome deficient mutants of P. denitrificans but expression and translocation of an apocytochrome C550-alkaline phosphatase fusion protein and apocytochrome cd1 were unaffected in HN53. The results suggest that the substrate for the putative CcmABC-transporter is probably neither haem nor c-type apocytochromes.

ATP-Binding Cassette Transporters↗

PubMed1997

Expression of the mau gene cluster of Paracoccus denitrificans is controlled by MauR and a second transcription regulator.

The mau gene cluster of Paracoccus denitrificans constitutes 11 genes (10 are located in the transcriptional order mauFBEDACJGMN; the 11th, mauR, is located upstream and divergently transcribed from these genes) that encode a functional methylamine-oxidizing electron transport branch. The mauR gene encodes a LysR-type transcriptional activator essential for induction of the mau operon. In this study, the characteristics of that process were established. By using lacZ transcriptional fusions integrated into the genome of P. denitrificans, it was found that the expression of the mauR gene during growth on methylamine and/or succinate was not autoregulated, but proceeded at a low and constant level. The mauF promoter activity was shown to be controlled by MauR and a second transcriptional regulator. This activity was very high during growth on methylamine, low on succinate plus methylamine, and absent on succinate alone. MauR was overexpressed in Escherichia coli by using a T7 RNA polymerase expression system. Gel shift assays indicated that MauR binds to a 403 bp DNA fragment spanning the mauR-mauF promoter region. It is concluded from these results that the expression of the structural mau genes is dependent on MauR and its inducer, methylamine, as well as on another transcription factor. Both activators are required for high-level transcription from the mauF promoter. It is hypothesized that the two activators act synergistically to activate transcription: the effects of the two activators are not additive and either one alone activates the mauF promoter rather weakly.

Escherichia coli↗