title: Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori.
abstract: The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.
authors.forename: N J
authors.initials: NJ
authors.lastname: Hughes
authors.forename: P A
authors.initials: PA
authors.lastname: Chalk
authors.forename: C L
authors.initials: CL
authors.lastname: Clayton
authors.forename: D J
authors.initials: DJ
authors.lastname: Kelly
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism*; D005418:Flavodoxin / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism; D008795:Metronidazole / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011773:Pyruvates / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D001639:Bicarbonates; D005418:Flavodoxin; D011773:Pyruvates; D008795:Metronidazole; D002245:Carbon Dioxide; D001710:Biotin; D008291:Malate Dehydrogenase; C030574:malate dehydrogenase (decarboxylating); D007658:Ketone Oxidoreductases; C039443:pyruvate-flavodoxin oxidoreductase; D000103:Acetyl-CoA Carboxylase