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PubMed1988

Stable isotope evidence for the localisation of phenylalanine biosynthesis in the bacterium Paracoccus denitrificans.

Paracoccus denitrificans was grown on [6-13C]-glucose as the sole carbon source for growth and the extracts were fractionated and analysed by gas chromatography-mass spectrometry. The 13C-labelling pattern observed in phenylalanine indicated that the biosynthetic sequences of enzymes for phenylalanine production were unequally distributed within the cell and that there are at least 2 separate loci of phenylalanine biosynthesis. The principal locus of phenylalanine production was associated with the Entner-Doudoroff and/or the pentose phosphate pathways and it was responsible for producing 3/4 of the bacterium's phenylalanine. A second locus was associated with the G6 oxidation pathway and was responsible for producing the remaining 1/4 of the cell's phenylalanine.

Carbon Isotopes↗

PubMed1985

Mechanism and stereospecificity of the parabactin-mediated iron-transport system in Paracoccus denitrificans.

The mechanism by which Paracoccus denitrificans utilizes parabactin in its iron-transport apparatus is examined. The cellular uptake of 55Fe-parabactin, its enantiomer, as well as a large number of its homologues and analogues are measured. Furthermore, the ability of these catecholamide ligands to stimulate microbial growth is also determined. The results of these studies point out several structural boundary conditions which the microorganism sets when utilizing iron chelates.

Biological Transport, Active↗

PubMed1980

Purification and properties of thiamine pyrophosphokinase in Paracoccus denitrificans.

The existence of thiamine pyrophosphokinase [EC 2.7.6.2] in procaryotic cells was first demonstrated in Paracoccus denitrificans (J. Bacteriol, (1976) 126, 1030-1036). The enzyme was therefore purified from this organism to determine its molecular structure and properties. Thiamine pyrophosphokinase which was purified 620-fold from P. denitrificans showed a single band on both polyacrylamide and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the molecular weight in the latter case was calculated to be 23,000. Gel filtration analysis using Sephadex G-150 gave a molecular weight of 44,000, indicating that this enzyme contains at least two identical subunits. Although sedimentation equilibrium analysis gave a molecular weight of 96,000, indirect evidence suggests that the form having this molecular weight is an aggregate of the functional dimer. The activity of the purified enzyme required thiamine, ATP, and Mg2+, and the enzyme catalyzed thepyrophosphorylation of thiamine by ATP. Km values for thiamine and ATP were 10 microM and 0.38 mM, respectively. The activity was competitively inhibited by pyrithiamine, giving a Ki value of 19 microM. Oxythiamine and chloroethylthiamine were very weak inhibitors of the enzyme. The activity was also inhibited by the product, TPP.

Kinetics↗

PubMed1981

Reaction of oxygen with cytochrome c oxidase from Paracoccus denitrificans.

The reaction of reduced cytochrome c oxidase (EC 1.9.3.1) from Paracoccus denitrificans (American Type Culture Collection 13543) with dioxygen has been followed by laser flash photolysis of the CO derivative. In detergent-stabilized solutions the reaction showed at least two distinct kinetic components, the faster of which was oxygen concentration dependent and had a rate of approximately 60 X 10(6) M-1 s-1. The slower reaction was independent of oxygen concentration and had a rate of 9 X 10(2) s-1. These rates are about 1.5 times greater than comparable rates for ox heart oxidase reported by C. Greenwood and Q. H. Gibson (J. Biol. Chem. (1967) 242, 1782-1787). The kinetic components have markedly different optical spectra which agree precisely in form with those for ox heart enzyme (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787) but are shifted by 2 nm toward the red. In phospholipid vesicles, the spectral contribution of the faster component was augmented. The dissociation constant for CO at 20 degrees C is 1.6 microM, 6 times greater than for the ox heart enzyme. The bacterial enzyme binds one CO per 2 heme a. The enzyme has an absorption band at 830 nm in the oxidized form similar to that of the ox heart enzyme.

Carbon Monoxide↗

PubMed1982

The cytochrome c oxidase of Paracoccus denitrificans pumps protons in a reconstituted system.

The purified two-subunit cytochrome c oxidase of Paracoccus denitrificans was reconstituted into phospholipid vesicles having a high internal buffering capacity and exhibiting a respiratory control index greater than 6.6. With these proteoliposomes, pH changes of the suspending medium were monitored in response to reductant pulses in the presence of valinomycin and potassium. When reduced cytochrome c was added to allow for a limited number of turnovers (2-12), a net acidification of the extravesicular space could be observed. This apparent proton ejection by the vesicles was abolished by inhibition of the oxidase with azide, by bypassing the oxidase with ferricyanide, or by preventing charge compensation by omitting valinomycin. Addition of uncoupler led to an alkalinization, rather than an acidification, of the extravesicular space in response to reduced cytochrome c. We thus conclude that cytochrome c oxidase of P. denitrificans is a proton pump. Under the conditions described here, an apparent stoichiometry of 0.6 proton ejected/electron was obtained by extrapolation to zero turnovers.

Animals↗

PubMed1982

Solubilized cytochrome c oxidase from Paracoccus denitrificans is a monomer.

Cytochrome c oxidase purified from the bacterium Paracoccus denitrificans was analyzed by analytical ultracentrifugation. In the detergent octyltetra/pentaoxyethylene (C8E45), the isolated enzyme exhibits a molecular weight of 79,000 to 84,000. The detergent-solubilized enzyme is thus a monomer which contains one copy of each of the two subunits.

Detergents↗

PubMed1981

Energy coupling to K+ transport in Paracoccus denitrificans.

Paracoccus denitrificans requires potassium for normal growth and transports this cation by at least two systems, one with low (Km approximately 1 to 2 mM) and another with high (Km approximately 0.1 microM) affinity. Neither of the two systems seems to be dependent on periplasmic components since each retains full activity in cells subjected to an osmotic shock. P. denitrificans accumulates potassium at high velocity (270 nmol of K+/min/mg dry weight of cells) and against a large concentration gradient. The intracellular concentration of K+ in media of high osmolarity (about 320 mosmol) is 0.4 M; this gives a concentration gradient [K+]i/[K+]e of greater than or equal to 2 X 10(4). The uptake of potassium against its concentration gradient requires a source of energy and is eliminated by the addition of uncouplers. The increased rate of energy usage for potassium transport results in an increased rate of ATP synthesis by the respiratory chain and is expressed in enhanced rates of respiration and substrate utilization. The stimulation of respiration is accompanied by increased steady state reduction level of the components of the respiratory chain. The calculations show that two K+ are most likely to be transported per one ATP hydrolyzed.

Adenine Nucleotides↗

PubMed1995

Purification of Paracoccus denitrificans cytochrome c552 and sequence analysis of the gene.

Unlike mitochondria, many bacteria use a large repertoire of c-type cytochromes in different branches of their electron transport system. Among the many cytochromes c present in the soil bacterium Paracoccus denitrificans, a membrane-bound cytochrome (c552) has been suggested to mediate the electron transport between the cytochrome bc1 complex and cytochrome-c oxidase [Berry, E. A. & Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467]. We have purified this cytochrome from cytoplasmic membranes, and cloned and sequenced its gene, cycM. Sequence analysis reveals that, while its C-terminal portion is highly similar to type-I cytochromes c, its N-terminal part contains a hydrophobic segment providing membrane attachment. In addition, we present immunological evidence for its functional role in respiration.

Amino Acid Sequence↗

PubMed1994

Expression and characterization of human and chimeric human-Paracoccus denitrificans electron transfer flavoproteins.

Electron transfer flavoprotein (ETF) is a heterodimer that contains a single equivalent of FAD and accepts electrons from nine flavoprotein dehydrogenases in the mitochondrial matrix. Human ETF was expressed in Escherichia coli using the expression vector previously employed to express Paracoccus denitrificans ETF (Bedzyk, L. A., Escudero, K. W., Gill, R. E., Griffin, K. J., and Frerman, F. E. (1993) J. Biol. Chem. 268, 20211-20217). cDNAs encoding the beta and alpha subunits of the human protein were inserted into the vector, mimicking the arrangement of the P. denitrificans genes in which coding sequences are joined by overlapping termination and initiation codons. A human ETF containing 30% P. denitrificans sequence at the amino terminus of the beta subunit was also expressed and purified. This chimeric ETF has 64% sequence identity with the human sequence in the substituted region. Kinetic constants of medium chain and short chain acyl-CoA dehydrogenases for the chimeric ETFs were slightly changed from those of human ETF; but, there are marked differences in the kinetic constants of sarcosine dehydrogenase and electron transfer flavoprotein-ubiquinone oxidoreductase with the two ETFs. Absorption spectra of the three redox states of human, chimeric, and P. denitrificans ETF flavins are identical. However, the flavin circular dichroism spectra of the three ETFs are characteristic for each species. The spectrum of the chimeric ETF has both human and P. denitrificans ETF features. The amplitude of the 436 nm band is identical to that of the of the human ETF flavin, but the amplitude of the 375 nm band is identical to that of the P. denitrificans ETF flavin. Thus, flavin in the chimeric ETF appears to be exposed to dipoles in the protein framework provided by human and bacterial sequences. These spectral data indicate that the flavin is located in the vicinity of the amino-terminal region of the beta subunit. The kinetic data suggest that the amino-terminal region of the beta subunit comprises part of the docking site for some primary dehydrogenases and electron transfer flavoprotein-ubiquinone oxidoreductase.

Amino Acid Sequence↗

PubMed1994

Respiratory inhibitors activate an Fnr-like regulatory protein in Paracoccus denitrificans: implications for the regulation of the denitrification pathway.

Unlike the uncoupler carbonyl cyanide 3-chlorophenyl-hydrazone, the respiratory inhibitors CN-, N3-, NO2- and rotenone enhanced the formation of nitrate and nitrite reductases in highly aerated cultures of the Paracoccus denitrificans ex-conjugant PD1222 (pRW2A/FF). A maximal effect was observed at concentrations partly blocking electron transport to O2. The level of beta-galactosidase reporting the activity of an Fnr-like regulatory protein showed a similar concentration dependency. It is concluded that oxygen is sensed by Fnr in an indirect way, possibly via the redox state of a cellular component.

Aerobiosis↗

PubMed1994

Properties of the iron-sulfur center in the 25-kilodalton subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans.

The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans has been expressed in Escherichia coli and purified to homogeneity. EPR studies of the reduced recombinant protein indicated that the expressed subunit contains a single [2Fe-2S] cluster (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33, 494-499). In this report, the electronic, magnetic, and vibrational properties of the [2Fe-2S]2+,+ center have been investigated by the combination of absorption, circular dichroism, variable-temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopies and compared with a range of simple [2Fe-2S]-containing proteins. The results are consistent with coordination by two cysteinyl residues at both the reducible and nonreducible iron sites and reveal a striking similarity between the properties of the [2Fe-2S] cluster in the P. denitrificans NDH-1 25-kDa subunit and those of the subclass of ferredoxin-type [2Fe-2S] centers typified by Clostridium pasteurianum 2Fe ferredoxin. The four cyteines residues involved in cluster ligation in these proteins have been tentatively identified based on sequence homology considerations.

Amino Acid Sequence↗

PubMed1994

A cytochrome ba3 functions as a quinol oxidase in Paracoccus denitrificans. Purification, cloning, and sequence comparison.

A quinol oxidase has been purified from the cytoplasmic membrane of Paracoccus denitrificans; its heme composition and CO binding properties identify it as a cytochrome ba3. On SDS gels, the purified enzyme complex is separated into five polypeptides. Using partial peptide sequence information for subunit II, the gene locus has been cloned and sequenced. In a typical operon pattern, four genes were identified: qoxA, -B, -C, and -D, coding for subunits II, I, III, and IV. DNA-derived amino acid sequence comparisons reveal extensive similarities to other members of the terminal oxidase superfamily.

Amino Acid Sequence↗

PubMed1993

Soluble CuA-binding domain from the Paracoccus cytochrome c oxidase.

In cytochrome c oxidase the C-terminal part of subunit II is outside the membrane and contains a copper center called CuA. We have expressed this domain of the Paracoccus denitrificans oxidase in a soluble form. Data obtained by quantitative copper-to-protein measurements, electrospray mass spectrometry, and electron paramagnetic resonance spectroscopy show that the center contains two copper atoms probably in a mixed valence configuration. Its absorbance spectrum is similar to that of the copper center A in nitrous oxide reductase. The EPR spectrum suggests that the center in the soluble protein is closely related to the native CuA site in the cytochrome oxidase complex. However, it seems likely that the copper center in the soluble domain is more exposed to the aqueous milieu than in the intact complex because its absorbance and EPR spectra are sensitive to pH. At alkaline pH one of the coppers in the site acquires type-2 character, indicating that it may be coordinated to a new ligand. The pK of this reversible change is about 8.2. The CuA-binding fragment is able to oxidize cytochrome c.

Amino Acid Sequence↗

PubMed2001

Multivariable control of alcohol concentrations in the production of polyhydroxyalkanoates (PHAs) by Paracoccus denitrificans.

A novel multivariable control strategy is developed for alcohol (ethanol and n-pentanol) concentrations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(HB-co-HV), a biodegradable polymer by Paracoccus denitrificans ATCC 1774. This controller, which is developed to control the mole fraction of P(HB-co-HV), consists of two parts: one is for ethanol concentration control and the other is for mole fraction control, based on the concept of metabolic flux distribution control. A simple metabolic reaction (MR) model is constructed for flux distribution analysis. The relationship between mole ratio of specific consumption rate of the two alcohols (ethanol and n-pentanol) and the mole fraction of 3HV units in the polymer is linear. This result suggests that the split ratio at a branched point of 3-ketovaleryl-CoA in the P(HB-co-HV) synthetic pathway is constant for several fermentation conditions. When the mole fraction of 3HV units has a target value, the feed rate of n-pentanol becomes a function of the feed rate of ethanol and the set value of 3HV, based on the MR model. The mole fraction of 3HV units successfully reached the target value using this strategy. The mole fraction control strategy is combined with an optimal production strategy based on the optimal profile of the specific growth rate. The combined strategy is realized using multivariable controllers and P(3HB-co-3HV) production is maximized with a given value of mole fraction of 3HV units at the final step of fermentation.

Acyl Coenzyme A↗

PubMed1997

Complete nucleotide sequence of the replicator region of Paracoccus (Thiobacillus) versutus pTAV1 plasmid and its correlation to several plasmids of Agrobacterium and Rhizobium species.

The complete nucleotide sequence of the replicator region of pTAV1, a cryptic, low copy number plasmid of Paracoccus versutus, was determined. The minimal replicon sequence (3149 bp) included in pTAV203/18 contains two open reading frames with coding capabilities for putative polypeptides of 23.8 (RepX) and 46 kDa (RepC'). The two genes have the same transcriptional polarity and both seem to be essential for replication of pTAV203. The predicted amino acid sequence of RepC' shows significant homology with the major replication-associated proteins of several Agrobacterium and Rhizobium plasmids. A probable origin of replication (oriV) was proposed to be localized at the 3' terminal end of the repC' gene.

Amino Acid Sequence↗

PubMed1997

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Omega) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases.

Journal Article↗

PubMed1998

Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes.

Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I-IV is obtained when the purification procedure is performed in beta-d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I-II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

Journal Article↗

PubMed2000

Transcriptional regulation of nir and nor operons of Paracoccus denitrificans.

Transcripts of nitrite reductase (nir) and nitric oxide reductase (nor) operons of Paracoccus denitrificans were expressed only under anaerobic conditions in the presence of potassium nitrite. The nir and nor operons produced at least two transcription products. Large transcripts seemed to contain whole operons and small ones seemed to contain the genes encoding denitrifying enzymes. All transcription start sites were detected 41.5 or 42.5 bp downstream from the center of FNR boxes.

Journal Article↗