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PubMed2001

The cytochrome c domain of dimeric cytochrome cd(1) of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli.

Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre. The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69. Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here. The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1).

Cloning, Molecular↗

PubMed1998

Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans has been refined at 1.75 A resolution utilizing the DNA-based protein sequence. The final model incorporates 8034 atoms per molecule, including 552 molecules of solvent, and gives an R-factor of 0.163. The molecule is an H2L2 hetero-tetramer containing a non-crystallographic 2-fold axis of symmetry. The 373-residue H subunit is folded into seven repeats of a four-stranded antiparallel beta-sheet motif, arranged in a propeller-like pattern about a pseudo-7-fold rotational axis of symmetry. Each L subunit contains 131 residues folded in a tight structure composed of five beta-strands in two sheets and crosslinked by six disulfide bonds. In addition there is an intrasubunit covalent linkage between two tryptophan side-chains that form the unique redox center, tryptophan tryptophylquinone (TTQ). The active site contains the O-6 carbonyl of TTQ, the side-chains of Asp32L Asp76L, Tyr119L and Thr122L, and two solvent molecules. A potential "gate" (Phe55H) separates the closed active-site cavity from a channel containing a group of highly ordered water molecules to bulk solvent. Phe55H and Tyr119L, and a number of neighboring oxygen atoms, may also provide a binding site for monovalent cations that are known to affect the reactivity and spectral properties of TTQ as well as the oxidative half reaction. The overall reaction has been dissected into a number of discrete steps that may require participation by several individual amino acid residues in the active site acting as general acids and bases.

Amino Acid Sequence↗

PubMed1998

Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network.

Aminotransferase reversibly catalyzes the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5'-phosphate (PLP) as a cofactor. Various kinds of aminotransferases developing into catalysts for particular substrates have been reported. Among the aminotransferases, aromatic amino acid aminotransferase (EC 2.6.1. 57) catalyzes the transamination reaction with both acidic substrates and aromatic substrates. To elucidate the multiple substrate recognition mechanism, we determined the crystal structures of aromatic amino acid aminotransferase from Paracoccus denitrificans (pdAroAT): unliganded pdAroAT, pdAroAT in a complex with maleate as an acidic substrate analog, and pdAroAT in a complex with 3-phenylpropionate as an aromatic substrate analog at 2.33 A, 2. 50 A and 2.30 A resolution, respectively. The pdAroAT molecule is a homo-dimer. Each subunit has 394 amino acids and one PLP and is divided into small and large domains. The overall structure of pdAroAT is essentially identical to that of aspartate aminotransferase (AspAT) which catalyzes the transamination reaction with only an acidic amino acid. On binding the acidic substrate analog, arginine 292 and 386 form end-on salt bridges with carboxylates of the analog. Furthermore, binding of the substrate induces the domain movement to close the active site. The recognition mechanism for the acidic substrate analog in pdAroAT is identical to that observed in AspAT. Binding of the aromatic substrate analog causes reorientation of the side-chain of the residues, lysine 16, asparagine 142, arginine 292* and serine 296*, and changes in the position of water molecules in the active site to form a new hydrogen bond network in contrast to the active site structure of pdAroAT in the complex with an acidic substrate analog. Consequently, the rearrangement of the hydrogen bond network can form recognition sites for both acidic and aromatic side-chains of the substrate without a conformational change in the backbone structure in pdAroAT.

Bacterial Proteins↗

PubMed2000

Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states.

The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1. 4 A for the reduced state. This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states. The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes. In general, the structure has the same features as that of known eukaryotic cytochromes c. However, the surface properties are very different. Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms. One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c. It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A. Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c. The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one.

Crystallography, X-Ray↗

PubMed2001

Crystal structure of chaperonin-60 from Paracoccus denitrificans.

The crystal structure of chaperonin-60 from Paracoccus denitrificans (P.cpn60) has been determined at 3.2 A resolution by the molecular replacement method. Two heptameric rings of identical subunits of P.cpn60 in adjacent asymmetric units are stacked in a back-to-back manner and form a cylinder, as found in GroEL, cpn60 from Escherichia coli. With respect to the unliganded GroEL structure, each subunit of P.cpn60 tilts 2 degrees outwards and the apical domain twists 4 degrees counter-clockwise in the top view in a hinge-like manner, rendering the central hole 5 A wider. Despite the subunit tilts, both rings in P.cpn60 contact at two sites of the equatorial domain in the same way as in GroEL. Interactions between residues 434 and 434, and 463 and 463 observed in GroEL were not found in P.cpn60, and the interaction between 452 and 461 was weaker in P.cpn60 than in GroEL. The unique hydrogen bond between 468 and 471 was observed at the right site in P.cpn60, which could account for why the subunits tilt outwards. The contact surface area was reduced at the left site, which is similar to the observed changes in the GroEL structures induced by ATP binding. In general, inter-ring interactions in P.cpn60 were weakened, which is consistent with findings that P.cpn60 is observed in single-ring forms as well as in double-ring forms.

Amino Acid Sequence↗

PubMed1995

Isolation and characterization of a novel insertion sequence element, IS1248, in Paracoccus denitrificans.

A new suicide vector, pRVS3, was constructed to facilitate gene replacements in the genome of Paracoccus denitrificans. In control experiments, incorporation of this suicide vector into the genome did not depend on the presence of homologous DNA. Using appropriate restriction enzymes, the suicide vector and flanking DNA were recovered from the genomic DNA. Sequence analysis demonstrated that both up- and downstream of the ex-integrant vector there was an element that showed high homology with bacterial insertion sequences (IS). Southern blot analysis of wild-type and integrant strains revealed that at least four copies of this IS element reside in the P. denitrificans genome, one of which, designated IS1248, had been involved in the transpositional event described here. IS1248 is 830 bp long, has 13-bp imperfect inverted repeats at the borders, and contains five open reading frames. With respect to the organization and primary sequences of the open reading frames, IS1248 closely resembles IS869 and IS427 of Agrobacterium tumefaciens, IS402 of Pseudomonas cepacia, and ISmyco found in Mycobacterium tuberculosis.

Amino Acid Sequence↗

PubMed1979

Structural analysis of four strains of Paracoccus denitrificans.

Two out of eleven newly isolated strains of Paracoccus denitrificans were investigated by light and electron microscopic methods and compared with two strains of P. denitrificans already kept in culture collections. Samples were taken from different growth phases revealing short rods and nearly spherical cells in the exponential growth phase, and an increasing ratio of nearly spherical cells in the stationary growth phase. Cell division followed the binary fission mode; higher cell aggregates were not observed. Fine structural analysis revealed extracellular surface material stainable with Ruthenium red, a gram-negative cell wall and different storage material inclusions. Structural properties and variations within the four strains under investigation are discussed and compared with those of related bacteria.

Cell Division↗

PubMed1984

The effect of oxygen on denitrification during steady-state growth of Paracoccus halodenitrificans.

Steady-state cultures of Paracoccus halodenitrificans were grown anaerobically prior to establishing steady states at different concentrations of oxygen. In the absence of oxygen, nitrate-limited cultures produced dinitrogen, and as the oxygen supply increased, these cultures produced nitrous oxide, then nitrite. These changes reflected two phenomena: the inactivation of nitrous oxide reductase by oxygen and the diversion of electrons from nitrite to oxygen.

Aerobiosis↗

PubMed1984

A dissimilatory nitrite reductase in Paracoccus halodenitrificans.

Paracoccus halodenitrificans produced a membrane-associated nitrite reductase. Spectrophotometric analysis showed it to be associated with a cd-cytochrome and located on the inner side of the cytoplasmic membrane. When supplied with nitrite, membrane preparations produced nitrous oxide and nitric oxide in different ratios depending on the electron donor employed. The nitrite reductase was maximally active at relatively low concentrations of sodium chloride and remained attached to the membranes at 100 mM sodium chloride.

Cell Membrane↗

PubMed1977

Metabolic regulation of pyruvate kinase isolated from autotrophically and heterotrophically grown Paracoccus denitrificans.

Pyruvate kinase (ATP: pyruvate phosphotransferase (EC 2.7.1.40) was partially purified from both autotrophically and heterotrophically grown Paracoccus denitrificans. The organism grown under heterotrophic conditions contains four times more pyruvate kinase than under autotrophic conditions. The enzyme isolated from both sources exhibited sigmoidal kinetics for both phosphoenolpyruvate (PEP) and ADP. The apparent Km for ADP and PEP in the "autotrophic" enzyme were 0.63 mM ADP and 0.25 mM PEP. The effect of several low molecular weight metabolites on the pyruvate kinase activity was investigated. Ribose-5-phosphate, glucose-6-phosphate and AMP stimulated the reaction at low ADP levels; this stimulation was brought about by an alteration in the apparent Km for ADP. The pyruvate kinases differ in their response to adenine nucleotides, but both preparations seem to be under adenylate control. The results are discussed in relation to the role of pyruvate kinase as a regulatory enzyme in P. denitrificans grown under both autotrophic and heterotrophic conditions.

Adenosine Monophosphate↗

PubMed1977

Metabolic regulation of the glucose-6-phosphate dehydrogenase from Paracoccus denitrifcans grown on glucose/nitrate.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate : NADP+ L-oxidoreductase EC 1.1.1.49) isolated from Paracoccus denitrificans grown on glucose/nitrate exhibits both NAD+-and NADP+- linked activities. Both activities have a pH optimum of pH 9.6 (Glycine/NaOH buffer) and neither demonstrates a Mg2+ requirement. Kinetics for both NAD(P)+ and glucose-6-phosphate were investigated. Phosphoenolpyruvate inhibits both activities in a competitive manner with respect to glucose-6-phosphate. ATP inhibits the NAD+-linked activity competitively with respect to glucose-6-phosphate but has no effect on the NADP+-linked activity. Neither of the two activities are inhibited by 100 muM NADH but both are inhibited by NADPH. The NAD+-linked activity is far more sensitive to inhibition by NADPH than the NADP+-linked activity.

Adenosine Triphosphate↗

PubMed1980

The regulation of hydrogenase formation as a differentiating character of strains of Paracoccus denitrificans.

Paracoccus denitrificans strains Stanier 381 (DSM 65), Morris (DSM 413), and Vogt 11 (DSM 415) and eleven newly isolated strains were compared with respect to the localization of hydrogenase and its regulation. In all strains hydrogenase was found to be membrane-bound and not able to reduce pyridine nucleotides. The enzyme was inducible in strain 381 and was found only in cells grown with hydrogen as the sole hydrogen donor; in cells grown under mixotrophic or heterotrophic conditions the hydrogenase activity was zero. In all other strains hydrogenase was constitutive and was present in cells grown under autotrophic, mixotrophic and heterotrophic conditions. Under the latter conditions the specific hydrogenase activity was even higher than under mixotrophic conditions.

Aerobiosis↗

PubMed1985

Statistical analysis of growth limitations in Paracoccus denitrificans: an experiment with a completely randomized two-way factorial design with replications.

Cultivation of microorganisms under growth limitation is a widely used technique in microbiology. The relevant investigations, though seemingly based on straightforward experiments, have generated conflicting results, e.g., concerning molar growth yields. The purpose of this paper is to show that discrepancies in the literature could be solved if more attention were paid to methodology, especially statistics. New experimental results, concerning growth limitations in Paracoccus denitrificans, will exemplify this. They include the following items. Two limitations, one of them being succinate limitation, were established in the present study. Molar growth yields on succinate were 41 g X mol-1 for succinate limitation (95% confidence limits were 38 and 44) and 32 g X mol-1 for the other limitation (95% confidence limits were 29 and 35). The latter result is compatible with sulphate limitation, but the present experimental design does not really permit this conclusion.

Analysis of Variance↗

PubMed1977

Energy conservation during aerobic growth in Paracoccus denitrificans.

Paracoccus denitrificans was aerobically grown in chemostat culture with succinate or gluconate as carbon source. Due to the presence of two phosphorylation sites in the respiratory chain and the absence of branching, theoretical P/O ratios of 1.71 and 1.82 were calculated for cells growing respectively with succinate and gluconate as carbon source. Using these data, 95% confidence intervals for the P/O ratio were determined, via a mathematical model, at 0.91-1.15 and 1.00-1.37 for sulphate-limited cultures, with respectively succinate and gluconate as carbon source. These results and measurements of P/O ratios in membrane particles and of proton translocation in whole cells have led to the conclusion that site I phosphorylation is affected under sulphate-limited conditions. Under conditions of carbon source-limitation the endogenous leads to H+/O ratio is about 7-8. Average values of 3.40 and 4.78 were respectively found for sulphate-limited succinate- and gluconate grown cells. For starved cells, oxidizing succinate as exogenous substrate, the leads to H+/O ratios were determined at about 3-4, independent of the growth limiting factor. It is concluded that the number of protons ejected per pair of electrons per energy-conserving site (leads to H+/site ratio) is about 3-4, instead of 2 as postulated by the chemiosmotic hypothesis.

Aerobiosis↗

PubMed1986

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans.

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membrane-bound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membrane-bound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.

Ammonia↗

PubMed1980

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans.

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide Mr = 150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrance protein Mr = 150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are co-regulated and that the active enzyme has a role in regulating its own synthesis.

Azides↗

PubMed1980

Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans.

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein Mr150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutuant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein Mr150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wild type. Autoradiography of preparations from cells incubated with 55Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with 99Mo, incorporation of molybdenum into the mutant protein was not detectable. We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.

Aldehyde Oxidoreductases↗

PubMed1992

Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes.

Denitrification and methylotrophy in Paracoccus denitrificans are discussed. The properties of the enzymes of denitrification: the nitrate-nitrite antiporter, nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase are described. The genes for none of these proteins have yet been cloned and sequenced from P. denitrificans. A number of sequences are available for enzymes from Escherichia coli, Pseudomonas stutzeri and Pseudomonas aeruginosa. It is concluded that pathway specific c-type cytochromes are involved in denitrification. At least 40 genes are involved in denitrification. In methanol oxidation at least 20 genes are involved. In this case too pathway specific c-type cytochromes are involved. The sequence homology between the quinoproteins methanol dehydrogenase, alcoholde-hydrogenase and glucose dehydrogenase is discussed. This superfamily of proteins is believed to be derived from a common ancestor. The moxFJGI operon determines the structural components of methanol dehydrogenase and the associated c-type cytochrome. Upstream of this operon 3 regulatory proteins were found. The moxY protein shows the general features of a sensor protein and the moxX protein those of a regulatory protein. Thus a two component regulatory system is involved in both denitrification and methylotrophy. The phylogeny of prokaryotes based on 16S rRNA sequence is discussed. It is remarkable that the 16S rRNA of Thiosphaera pantotropha is identical to that of P. denitrificans. Still these bacteria show a number of differences. T. pantotropha is able to denitrify under aerobic circumstances and it shows heterotrophic nitrification. Nitrification and heterotrophic nitrification are found in species belonging to the beta-and gamma-subdivisions of purple non-sulfur bacteria. Thus the occurrence of heterotrophic nitrification in T. pantotropha, which belongs to the alpha-subdivision of purple non-sulfur bacteria is a remarkable property. Furthermore T. pantotropha contains two nitrate reductases of which the periplasmic one is supposed to be involved in aerobic denitrification. The nitrite reductase is of the Cu-type and not of the cytochrome cd1 type as in P. denitrificans. Also the cytochrome b of the Qbc complex of T. pantotropha is highly similar to its counterpart in P. denitrificans. It is hypothesized that the differences between these two organisms which both contain large megaplasmids is due to a combination of loss of genetic information and plasmid-coded properties. The distribution of a number of complex metabolic systems in eubacteria and in a number of species belonging to the alpha-group of purple non sulphur bacteria is reviewed.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗