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PubMed2000

Purification, crystallization and preliminary crystallographic studies of an integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli.

Cytochrome bo(3) ubiquinol oxidase has been successfully purified for crystallization. Single crystals of this integral membrane protein diffract X-rays to 3.5 A resolution and belong to the orthorhombic space group C222(1). From the diffraction data, the unit-cell parameters were determined to be a = 91.3, b = 370.3, c = 232.4 A. The crystals have a solvent content of 59% and contain two molecules per asymmetric unit. A search model generated from the structures of cytochrome c oxidase from Paracoccus denitrificans and the extrinsic domain of cytochrome bo(3) ubiquinol oxidase from Escherichia coli was used for molecular-replacement studies, resulting in a solution with sensible molecular packing.

Crystallization↗
PubMed2003

1.3 A X-ray structure of an antibody Fv fragment used for induced membrane-protein crystallization.

The antibody Fv fragment 7E2 has previously been employed in the induced crystallization of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans. The 1.3 A X-ray structure of the uncomplexed antibody fragment reveals conserved water networks on the surfaces of the framework regions. A novel consensus motif for water coordination, XX(S/T), is found along the edges of the beta-sandwich, where a water molecule forms hydrogen bonds to the carbonyl O atom of a residue at position N and the OG hydroxyl groups of conserved serines or threonines at position N + 2. Multiple conformations were found in the hydrophobic core for residues IleL21, LeuL33 and the disulfide bridges. An internal water molecule that is compatible with only one of the three packing states of the V(L) core suggests local 'breathing' of the variable domain. TrpH47, a conserved key residue of the V(H)/V(L) interface, is crucially involved in the formation of the antigen-binding site by adopting a novel conformation that specifically stabilizes the non-canonical CDR-L3 loop. Finally, a comparison with 7E2-cytochrome c oxidase complexes demonstrates that binding of this membrane-bound antigen proceeds without major conformational changes of the 7E2 antibody fragment.

Antibodies↗
PubMed2000

Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus.

The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).

Amino Acid Sequence↗
PubMed1993

Gene transfer of a part of a beta-lactamase gene?

beta-Lactamase is an enzyme which catalyzes the hydrolysis of the beta-lactam ring of penicillins and cephalosporins. By similarity analysis of amino acid sequences in a database, the amino acid sequence deduced from the nucleotide sequence of the upstream region of cytochrome c oxidase subunit II from Paracoccus denitrificans was found to have an unusually high score of homology to that of a portion of beta-lactamases from Gram-negative bacteria. Furthermore, the nucleotide sequences corresponding only to this region had a very high score of similarity among them. The phylogenetic tree constructed on the basis of the amino acid sequences was in accord with that constituted on the 5S rRNA's. Moreover, the molar G + C contents and the codon usage were similar to those in their respective bacteria. It is suggested, therefore, that the nucleotide sequence in P. denitrificans was positioned by a transfer of a part of a beta-lactamase gene formed as a result of gene duplication or it was formed by a deletion of the essential region of the beta-lactamase gene, although no beta-lactamase gene has been yet detected in P. denitrificans.

Amino Acid Sequence↗
PubMed1992

Cloning, sequencing and deletion from the chromosome of the gene encoding subunit I of the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides.

The ctaD gene encoding subunit I of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been cloned. The gene encodes a polypeptide of 565 residues which is highly homologous to the sequences of subunit I from other prokaryotic and eukaryotic sources, e.g. 51% identity with that from bovine, and 75% identity with that from Paracoccus denitrificans. The ctaD gene was deleted from the chromosome of R. sphaeroides, resulting in a strain that spectroscopically lacks cytochrome a. This strain maintains about 50% of the cytochrome c oxidase activity of the wild-type strain owing to the presence of an alternate o-type cytochrome c oxidase. The aa3-type oxidase was restored by complementing the chromosomal deletion with a plasmid-borne copy of the ctaD gene. This system is well suited for site-directed mutagenesis probing of the structure and function of cytochrome c oxidase.

Amino Acid Sequence↗
PubMed2005

Impact of the bacterial type I cytochrome c maturation system on different biological processes.

In the alpha-, beta- and gamma-Proteobacteria, the so-called cytochrome c maturation (Ccm) system is known to promote the covalent attachment of the haem to periplasmic apocytochrome c. However, in species of Pseudomonas, Rhizobium, Paracoccus and Legionella, mutations in ccm genes result in phenotypes that cannot be readily explained by the simple loss of a c-type cytochrome. These phenotypes include loss of siderophore production and utilization, reduced abilities to grow in low-iron conditions and in mammalian and protozoan host cells, and alterations in copper sensitivity and manganese oxidation. These various data suggest that Ccm proteins may perform one or more functions in addition to Ccm, which are critical for bacterial physiology and growth. Novel hypotheses that should be explored include the utilization of Ccm-associated haem for processes besides attachment to apocytochrome c, the export of a non-haem compound through the Ccm system, and the negative effects of protoporphyrin IX accumulation.

Cytochrome c Group↗
PubMed1982

Purification and characterization of the cytochrome c oxidase from Rhodopseudomonas sphaeroides.

When grown aerobically in the dark, Rhodopseudomonas sphaeroides develops a respiratory chain similar to that in mitochondria and the photosynthetic apparatus is suppressed. The aa3-type cytochrome c oxidase from Rps. sphaeroides has been purified in Triton X-100 by affinity chromatography with Sepharose 4B coupled to yeast cytochrome c. The oxidase contains 14 nmol heme a/mg protein and is composed of three polypeptide subunits with relative molecular masses of 45000, 37000 and 35000. The enzyme is highly active in the presence of detergents, with a maximal velocity of 300 s-1/mol oxidase using either yeast or horse-heart cytochrome c. The Rps. sphaeroides oxidase is cross-reactive with antibodies directed against the oxidases from Paracoccus denitrificans and Saccharomyces cerevisiae. A particularly close relationship is indicated in the case of P. denitrificans. The Rps. sphaeroides oxidase has been incorporated into phospholipid vesicles. The resulting oxidase in these vesicles demonstrates high enzymatic activity and a respiratory control ratio of 5. Using these vesicles, no evidence for proton extrusion accompanying cytochrome c oxidation was observed. The data suggest that the Rps. sphaeroides oxidase does not function as a proton pump.

Bacterial Proteins↗
PubMed1991

Cytochrome c2 is essential for electron transfer to nitrous oxide reductase from physiological substrates in Rhodobacter capsulatus and can act as an electron donor to the reductase in vitro. Correlation with photoinhibition studies.

1. Addition of nitrous oxide to a periplasmic fraction released from Rhodobacter capsulatus strains MT1131, N22DNAR+ or AD2 caused oxidation of c-type cytochrome, as judged by the decrease in absorbance at 550 nm. The periplasmic fraction catalysed reduction of nitrous oxide in the presence of either isoascorbate plus phenazine ethosulphate or reduced methyl viologen. The rates with these two electron donors were similar and were comparable to the activity observed with a quantity of cells equivalent to those from which the periplasm sample had been derived. Activity in the periplasm could not be observed with ascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine although this reductant was effective with intact cells treated with myxothiazol to block the activity of the cytochrome-bc1 complex. 2. Cells of R. capsulatus MTG4/S4, a mutant from which the gene for cytochrome c2 has been specifically deleted, did not catalyse detectable rates of nitrous-oxide reduction. A nitrous-oxide reductase activity was present, as shown by activity of both cells and a periplasmic fraction with isoascorbate plus phenazine ethosulphate as reductant. The rates in cells and the periplasmic fraction were similar to those observed in the corresponding wild-type strain (MT1131). In contrast to wild-type cells, 2,3,5,6-tetramethyl-p-phenylenediamine and N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2] were ineffective as mediators of electrons from isoascorbate. Visible absorption spectra showed that no detectable cytochromes in either the periplasm or intact cells of the MTG4/S4 mutant were oxidised by nitrous oxide. 3. Purified ferroycytochrome c2 from R. capsulatus was oxidised by nitrous oxide in the presence of periplasm from R. capsulatus MTG4/S4. The rate of oxidation was proportional to the amount of periplasm added, but was considerably lower than the rate of nitrous-oxide reduction observed with the same periplasmic fraction when either ascorbate plus phenazine ethosulphate or reduced methyl viologen were used as substrates. The oxidation of cytochrome c2 was inhibited by acetylene and by low concentrations of NaCl. 4. Oxidation of ferrocytochrome c2 by nitrous oxide was observed when the purified cytochrome was mixed with a preparation of nitrous-oxide reductase. However, oxidation of ferrocytochrome c' by nitrous oxide was not observed in the presence of the reductase. The observations with the mutant MTG4/S4 suggest that cytochrome c2 is the only periplasmic cytochrome involved in nitrous-oxide reduction. 5. Nitrous-oxide-dependent oxidation of a c-type cytochrome was observed in a periplasmic fraction from Paracoccus denitrificans, provided the fraction was first reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome c Group↗
PubMed1993

cDNA cloning and mitochondrial import of the beta-subunit of the human electron-transfer flavoprotein.

We have isolated a cDNA clone which encodes the entire beta-subunit of human electron-transferring flavoprotein (ETF) by screening an expression library from human liver using polyclonal antibodies against porcine ETF. This cDNA encodes a protein of 255 amino-acid residues with a predicted molecular mass of 27,877 Da which shows a high degree of similarity with partial amino-acid sequences obtained from both rat liver and Paracoccus denitrificans beta-ETF. Northern-blot analysis shows that the human beta-ETF mRNA is approximately 1 kb in size and is abundant in liver, heart and skeletal muscle. Incubation with intact mitochondria indicates that the cDNA-encoded beta-ETF polypeptide contains the information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that beta-ETF, unlike the majority of nuclear-encoded mitochondrial matrix proteins, does not have a cleavable leader peptide. Furthermore, when valinomycin is added to the incubation mixture, the import is abolished, thus demonstrating that it is an energy-dependent process. Interestingly, the sequence analysis of beta-ETF protein identifies a 26.3% identity with the Fix A gene product of the nitrogen-fixing bacterium Azorhizobium caulinodans.

Amino Acid Sequence↗
PubMed1997

Protein and gene structure of the NADH-binding fragment of Rhodobacter capsulatus NADH:ubiquinone oxidoreductase.

Membranes of aerobically grown Rhodobacter capsulatus contain only one type of NADH:ubiquinone oxidoreductase which is homologous to the proton-translocating complex I. The K(m) value of the enzyme for NADH was determined to be 8 microM. After solubilization of the membranes with an alkylglucoside detergent, two fragments of complex I with molecular masses of 110 kDa and 140 kDa were isolated by chromatographic steps in the presence of detergent. Both fragments contain at least two polypeptides with apparent molecular masses of 46 kDa and 42 kDa. FMN was identified as cofactor in the preparations. Degenerative oligonucleotide primers were used to amplify a part of the sequence coding for the NADH-binding subunit of complex I by PCR. With the PCR product as probe, a genomic fragment was cloned and sequenced containing the genes encoding the two purified polypeptides and additional reading frames. The two genes are named nuoE and nuoF and are homologous to nqo2 and nqo1 of Paracoccus denitrificans. However, NuoE contains a C-terminal extension of 149 amino acids compared with Nqo2. NuoE and NuoF have molecular masses of 41259 Da and 47133 Da and contain the NADH-, FMN- and FeS-cluster-binding motifs.

Amino Acid Sequence↗
PubMedDate not supplied

Different lipid A types in lipopolysaccharides of phototrophic and related non-phototrophic bacteria.

Lipid A analyses confirm not only the present taxa of the purple nonsulfur bacteria (formerly Rhodospirillaceae), but also phylogenetical relatedness of distinct phototrophic to distinct non-phototrophic bacteria, as was suggested by cataloguing 16S rRNA. For example, lipid A with ester-bound 3-OH-10:0 and the rare amide-linked 3-oxo-14:0 is common to the phototrophic Rhodobacter capsulatus and Rhodobacter sphaeroides and also to Paracoccus denitrificans and Thiobacillus versutus. 'Lipid ADAG' (lipid A with 2,3-diamino-D-glucose (DAG)) occurs in the phototrophic Rhodopseudomonas viridis and Rhodopseudomonas palustris and also in the related non-phototrophic species, e.g., Nitrobacter winogradskyi, Pseudomonas diminuta, or Thiobacillus ferrooxidans. The phylogenetically more coherent purple sulfur bacteria (Chromatiaceae) uniformly contain D-mannose in their phosphate-free lipid A. Among the green bacteria, only the Chlorobiaceae but not the likewise chlorosome-containing Chloroflexaceae contain lipopolysaccharide. Lipid ADAG from R. viridis is a structural analogue of a biosynthetic precursor (lipid X) of enterobacterial lipid A. Lipid A synthase from Salmonella accepts not only lipid X but also the synthetic di-N-acyl-2,3-diamino-D-glucose analogue as substrate (Raetz, C.R.H., unpublished results). More and more naturally occurring lipid A's with both, 2,3-diaminoglucose and glucosamine ('mixed' lipid A, with 2,3-diaminoglucose or glucosamine dominating) are being found. Newly recognized lipid A and lipid ADAG types might offer the possibility of differentially stimulating desired biological activities in animals without also having the undesired endotoxic activities. The non-toxic lipid A from Rhodopseudomonas viridis for example is able to stimulate prostaglandin secretion in peritoneal macrophages and can be used as an antagonist to the endotoxic shock caused by Salmonella lipopolysaccharide.

Bacteria↗
PubMed1990

Genetics of carbon metabolism in methylotrophic bacteria.

The application of genetic techniques to the methylotrophic bacteria has greatly enhanced studies of these important organisms. Two methylotrophic systems have been studied in some detail, the serine cycle for formaldehyde assimilation and the methanol oxidation system. In both cases, genes have been cloned and mapped in Methylobacterium species (facultative serine cycle methanol-utilizers). In addition, methanol oxidation genes have been studied in an autotrophic methanol-utilizer (Paracoccus denitrificans) and three methanotrophs (Methylosporovibrio methanica, Methylomonas albus and Methylomonas sp. A4). Although much remains to be learned in these systems, it is becoming clear that the order of C1 genes has been conserved to some extent in methylotrophic bacteria, and that many C1 genes are loosely clustered on the chromosome. Operons appear to be rare, but some examples have been observed. The extension of genetic approaches to both the obligate and facultative methylotrophs holds much promise for the future in understanding and manipulating the activities of these bacteria.

Base Sequence↗
PubMed1996

Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria.

Fifty different polyhydroxyalkanoic acid (PHA)-accumulating bacterial strains were investigated for the occurrence of phasin proteins bound to PHA granules and related to the GA24 protein of Alcaligenes eutrophus H16, by isolating PHA granules and Western blot analysis of granule-associated proteins employing antibodies raised against the GA24 protein. It could be demonstrated that th PHA granules of many poly(3-hydroxybutyrate)-accumulating bacteria exhibited a similar protein pattern, and a predominant protein of 24 +/- 2 kDa occurred in the granules of A. eutrophus strains A7, CH34, JMP222, N9A and TF93 exhibiting N-terminal amino acid sequences identical to that of the GA24 protein. Proteins bound to the granules of A. latus, Burkholderia caryophyli, B. cepacia, B. solanacearum, Pseudomonas glathei, Rhodobacter sphaeroides and Telluria mixta also gave positive immunoreactions. Granule-associated proteins of small size also occurred in various strains of the Gram positive bacteria Bacillus megaterium and R. ruber as well as in the Gram-negative bacteria Azotobacter sp., Chromatium vinosum, Comamonas acidovorans, Methylobacterium sp., Mycoplana rubra, Paracoccus denitrificans, Pseudomonas sp., Rhodospirillum rubrum, Rubrivivax gelatinosus and Thiocystis violacea; however, they gave no immunoreaction. This study clearly demonstrated that phasins are wide-spread if not essential in PHA-accumulating bacteria.

Alcaligenes↗
PubMed1997

Cloning and analysis of methanol oxidation genes in the methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding the alpha-subunit of methanol dehydrogenase (mxaF) and its flanking region was isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. The deduced amino acid sequence of MxaF showed 80, 80, 74 and 66% identity with those of Methylobacterium extorquens AM1, M. organophilum XX, Paracoccus denitrificans and Methylophilus methylotrophus, respectively. The putative mxaF promoter sequence (-35 -AAAGACA-, -10 -TAGAA-) observed in other methylotrophs was not found in the region 5' of H. methylovorum GM2 mxaF. Downstream of mxaF, five open reading frames and one partial open reading frame were detected which had high identity with the genes mxaJ, mxaG, mxaI, mxaR, mxaS and mxaA. This indicated the existence of a mxaFJGIRSA gene cluster in H. methylovorum GM2, as previously observed in M. extorquens AM1.

Alcohol Oxidoreductases↗
PubMed1998

Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments.

Two PCR primer sets for the nitrous oxide reductase gene (nosZ) were developed. The initial primers were based on three sequences in GenBank and used to amplify nosZ from continental shelf sediments and from two denitrifiers in culture, Thiosphaera pantotropha and Pseudomonas denitrificans. Three unique marine sediment nosZ genes were identified and sequenced. The marine nosZ genes were most closely related to the nosZ genes of Paracoccus denitrificans or to Rhizobium meliloti. Alignment of all nosZ sequences currently available (n = 10) facilitated redesign of the PCR primers. Three new primer sets which amplify 1100 bp, 900 bp and 250 bp regions of the nosZ gene were designed and tested. The new primers robustly amplified nosZ fragments from samples in which the initial nosZ primers were only marginally successful.

Amino Acid Sequence↗
PubMed2000

Characterization of an Azospirillum brasilense Tn5 mutant with enhanced N(2) fixation: the effect of ORF280 on nifH expression.

Disruption of an open reading frame (ORF) of 840 bp (280 amino acids; ORF280) in an Azospirillum brasilense Tn5 mutant resulted in a pleiotrophic phenotype. Besides an enhanced N(2)-fixing capacity and altered expression pattern of a nifH-gusA fusion, growth on the charged polar amino acids glutamate and arginine was severely affected. ORF280, similar to previously identified ORFs present in Bradyrhizobium japonicum (ORF277), Paracoccus denitrificans (ORF278) and Rhodobacter capsulatus (ORF277), exhibits in its C-terminus a significant similarity with the recently defined family of universal stress proteins.

Amino Acid Sequence↗
PubMed2001

Phylogeny and distribution of the soxB gene among thiosulfate-oxidizing bacteria.

A PCR protocol for the detection of sulfur-oxidizing bacteria based on soxB genes that are essential for thiosulfate oxidation by sulfur-oxidizing bacteria of various phylogenetic groups which use the 'Paracoccus sulfur oxidation' pathway was developed. Five degenerate primers were used to specifically amplify fragments of soxB genes from different sulfur-oxidizing bacteria previously shown to oxidize thiosulfate. The PCR yielded a soxB fragment of approximately 1000 bp from most of the bacteria. Amino acid and nucleotide sequences of soxB from reference strains as well as from new isolates and environmental DNA from a hydrothermal vent habitat in the North Fiji Basin were compared and used to infer relationships of soxB between sulfur-oxidizing bacteria belonging to various 16S rDNA-based phylogenetic groups. Major phylogenetic lines derived from 16S rDNA were confirmed by soxB phylogeny. Thiosulfate-oxidizing green sulfur bacteria formed a coherent group by their soxB sequences. Likewise, clearly separated branches demonstrated the distant relationship of representatives of alpha-, beta-, and gamma-Proteobacteria including representative species of the former genus Thiobacillus (now Halothiobacillus - gamma-Proteobacteria, Thiobacillus - beta-Proteobacteria and Starkeya - alpha-Proteobacteria). This general picture emerged although apparent evidence for lateral transfer of the soxB gene is indicated and comparison of soxB phylogeny and 16S rDNA phylogeny points to the significance of this gene transfer in hydrothermal vent bacterial communities of the North Fiji Basin.

Cytochrome c Group↗
PubMed1977

Numerically dominant denitrifying bacteria from world soils.

Nineteen soils, three freshwater lake sediments, and oxidized poultry manure were examined to determine the dominant denitrifier populations. The samples, most shown or expected to support active denitrification, were from eight countries and included rice paddy, temperate agricultural, rain forest, organic, and waste-treated soils. Over 1,500 organisms that could grow anaerobically on nitrate agar were isolated. After purification, 146 denitrifiers were obtained, as verified by production of N(2) from NO(3) (-). These isolates were characterized by 52 properties appropriate for the Pseudomonas-Alcaligenes group. Numerical taxonomic procedures were used to group the isolates and compare them with nine known denitrifier species. The major group isolated was representative of Pseudonomas fluorescens biotype II. The second most prevalent group was representative of Alcaligenes. Other Pseudomonas species as well as members of the genus Flavobacterium, the latter previously not known to denitrify, also were identified. One-third of the isolates could not utilize glucose or other carbohydrates as sole carbon sources. Significantly, none of the numerically dominant denitrifiers we isolated resembled the most studied species: Pseudomonas denitrificans, Pseudomonas perfectomarinus, and Paracoccus denitrificans. Denitrification appears to be a property of a very diverse group of gram-negative, motile bacteria, as shown by the large number (22.6%) of ungrouped organisms. The diversity of denitrifiers from a given sample was usually high, with at least two groups present. Denitrifiers, nitrite accumulators, and organisms capable of anaerobic growth were present in the ratio of 0.20+/-0.23:0.81+/-0.23:1. There were few correlations between their numbers and the sample characteristics measured. However, the temperatures at which isolates could grow were significantly related to the temperatures of the environments from which they were isolated. Regression analysis revealed few relationships between physical parameters and bacterial types, save for the anaerobe numbers, in which 94% of the variance could be accounted for.

Anaerobiosis↗