Characterization of the respiratory chain of Helicobacter pylori (original) (raw)
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FEMS Microbiology Letters, 1996
The quinone and cytochrome components of the respiratory chain of the microaerophilic bacterium Helicobacter pylon' have been investigated. The major isoprenoid quinone was menaquinone-6, with traces of menaquinone-4; no methyl-substituted or unusual menaquinone species were found. Cell yield was highest after growth at 10% (v/v) oxygen and menaquinone levels (per dry cell mass) were maxima1 at 5-10% (v/v> oxygen. Helicobacter pylori cells and membranes contained band c-type cytochromes, but not terminal oxidases of the aor d-types, as judged by reduced minus oxidised difference spectra. Spectra consistent with the presence of a CO-binding terminal oxidase of the cytochrome bor o-type were obtained. The soluble fraction from disrupted cells also contained cytochrome c. There were no significant qualitative differences in the cytochrome complements of cells grown at oxygen concentrations in the range 2-15% (v/v) but putative oxidases were highest in cells grown at 510% (v/v) oxygen.
The tricarboxylic acid cycle of Helicobacter pylori
European Journal of Biochemistry, 1999
The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [ 1 H]-and [ 13 C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, a-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including a-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and aketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of a-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a g-aminobutyrate shunt, owing to the absence of both g-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.
Microbiology, 2003
Helicobacter pylori whole cells showed high rates of oxygen uptake with L-serine and L-proline as respiratory substrates, and somewhat lower rates with D-alanine and D-proline. These respiratory activities were inhibited by rotenone and antimycin A at low concentrations. Since pyruvate was produced from L-serine and D-and L-alanine in whole cells, the respiratory activities with these amino acids as substrates occurred via pyruvate. Whole cells showed 2,6-dichlorophenolindophenol (DCIP)-reducing activities with D-and L-proline and D-alanine as substrates, suggesting that hydrogen removed from these amino acids also participated in oxygen uptake by the whole cells. High amounts of L-proline, D-and L-alanine, and L-serine were present in H. pylori cells, and these amino acids also predominated in samples of human gastric juice. H. pylori seems to utilize D-and L-proline, D-alanine and L-serine as important energy sources in its habitat of the mucous layer of the stomach.
MicroReview The diverse antioxidant systems of Helicobacter pylori
The gastric pathogen Helicobacter pylori induces a strong inflammatory host response, yet the bacterium maintains long-term persistence in the host. H. pylori combats oxidative stress via a battery of diverse activities, some of which are unique or newly described. In addition to using the well-studied bacterial oxidative stress resistance enzymes superoxide dismutase and catalase, H. pylori depends on a family of peroxiredoxins (alkylhydroperoxide reductase, bacterioferritin co-migratory protein and a thiolperoxidase) that function to detoxify organic peroxides. Newly described antioxidant proteins include a soluble NADPH quinone reductase (MdaB) and an iron sequestering protein (NapA) that has dual roles -host inflammation stimulation and minimizing reactive oxygen species production within H. pylori. An H. pylori arginase attenuates host inflammation, a thioredoxin required as a reductant for many oxidative stress enzymes is also a chaperon, and some novel properties of KatA and AhpC were discovered. To repair oxidative DNA damage, H. pylori uses an endonuclease (Nth), DNA recombination pathways and a newly described type of bacterial MutS2 that specifically recognizes 8-oxoguanine. A methionine sulphoxide reductase (Msr) plays a role in reducing the overall oxidized protein content of the cell, although it specifically targets oxidized Met residues. H. pylori possess few stress regulator proteins, but the key roles of a ferric uptake regulator (Fur) and a post-transcriptional regulator CsrA in antioxidant protein expression are described. The roles of all of these antioxidant systems have been addressed by a targeted mutant analysis approach and almost all are shown to be important in host colonization. The described antioxidant systems in H. pylori are expected to be relevant to many bacterial-associated diseases, as genes for most of the enzymes carrying out the newly described roles are present in a number of pathogenic bacteria.
FEBS Journal, 2009
Helicobacter pylori. Resistance to this drug is common among clinical isolates and results from loss of function mutations in rdxA, which encodes an oxygen insensitive nitroreductase (NTR). The RdxA-associated MTZ-reductase activity of H. pylori is lost upon cell disruption. Here we provide a mechanistic explanation for this phenomenon. Under aerobic conditions, His 6 -tagged RdxA protein (purified from Escherichia coli), catalyzed NAD(P)H-dependent reductions of nitroaromatic and quinone substrates including: nitrofurazone, nitrofurantoin, furazolidone, CB1954, and 1,4 benzoquinone, but not MTZ. Unlike other NTRs, His 6 -RdxA exhibited potent NAD(P)H oxidase activity (k cat = 2.8 s −1 ) which suggested two possible explanations for the role of oxygen in MTZ reduction: 1) NAD(P)H oxidase activity promotes cellular hypoxia (nonspecific reduction of MTZ); and 2) molecular oxygen out-competes MTZ for reducing equivalents. The first hypothesis was eliminated upon finding that rdxA expression, while increasing MTZ toxicity in both E. coli and H. pylori constructs, did not increase paraquat toxicity even though both are of similar redox potential. The second hypothesis was confirmed by demonstrating NAD(P)H-dependent MTZ-reductase activity (apparent K m = 122 ± 58 μM, k cat = 0.24 s −1 ) under strictly anaerobic conditions. The MTZ reductase activity of RdxA was 60-times greater than for NfsB (E. coli NTR), but 10 times lower than the NADPH-oxidase activity. Whether molecular oxygen directly competes with MTZ or alters the redox state of the FMN cofactors is discussed.
Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori
Free Radical Biology and Medicine, 2004
Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O 2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O 2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori. D
Archives of Microbiology, 2000
The respiratory chain enzymes of microaerophilic bacteria should play a major role in their adaptation to growth at low oxygen tensions. The genes encoding the putative NADH:quinone reductases (NDH-1), the ubiquinol:cytochrome c oxidoreductases (bc 1 complex) and the terminal oxidases of the microaerophiles Campylobacter jejuni and Helicobacter pylori were analysed to identify structural elements that may be required for their unique energy metabolism. The gene clusters encoding NDH-1 in both C. jejuni and H. pylori lacked nuoE and nuoF, and in their place were genes encoding two unknown proteins. The NuoG subunit in these microaerophilic bacteria appeared to have an additional Fe-S cluster that is not present in NDH-1 from other organisms; but C. jejuni and H. pylori differed from each other in a cysteine-rich segment in this subunit, which is present in some but not all NDH-1. Both organisms lacked genes orthologous to those encoding NDH-2. The subunits of the bc 1 complex of both bacteria were similar, and the Rieske Fe-S and cytochrome b subunits had significant similarity to those of Paracoccus denitrificans and Rhodobacter capsulatus, well-studied bacterial bc 1 complexes. The composition of the terminal oxidases of C. jejuni and H. pylori was dif-ferent; both bacteria had cytochrome cbb 3 oxidases, but C. jejuni also contained a bd-type quinol oxidase. The primary structures of the major subunits of the cbb 3 -type (terminal) oxidase of C. jejuni and H. pylori indicated that they form a separate group within the cbb 3 protein family. The implications of the results for the function of the enzymes and their adaptation to microaerophilic growth are discussed.
A Novel Insight into the Oxidoreductase Activity of Helicobacter pylori HP0231 Protein
PLoS ONE, 2012
Background: The formation of a disulfide bond between two cysteine residues stabilizes protein structure. Although we now have a good understanding of the Escherichia coli disulfide formation system, the machineries at work in other bacteria, including pathogens, are poorly characterized. Thus, the objective of this work was to improve our understanding of the disulfide formation machinery of Helicobacter pylori, a leading cause of ulcers and a risk factor for stomach cancer worldwide.
d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637
Amino Acids, 2010
Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. D-Amino acid dehydrogenase is a flavoenzyme that digests free neutral D-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 D-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial D-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to D-proline. The enzyme mediated electron transport from D-proline to coenzyme Q 1 , thus distinguishing it from D-amino acid oxidase. The apparent K m and V max values were 40.2 mM and 25.0 lmol min -1 mg -1 , respectively, for dehydrogenation of D-proline, and were 8.2 lM and 12.3 lmol min -1 mg -1 , respectively, for reduction of Q 1 . The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian D-amino acid oxidase than other bacterial D-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from D-proline to a c-type cytochrome was suggested spectrophotometrically.