IDENTIFICATION OF CARBOXYLATION ENZYMES AND CHARACTERIZATION OF A NOVEL 4-SUBUNIT PYRUVATE-FLAVODOXIN OXIDOREDUCTASE FROM HELICOBACTER-PYLORI

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by (HCO3-)-C-14 fixation and spectrophotometric assays. Acetyl coenzy me A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activitie s were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoeno lpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykina se (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent , ATP-independent, and avidin-insensitive (HCO3-)-C-14 fixation activi ty, which was shown to be due to the isotope exchange reaction of pyru vate: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-term inal sequence analysis showed that this enzyme is related to a recentl y recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pyl ori was found to mediate the reduction of a number of artificial elect ron accepters in addition to a flavodoxin isolated from H. pylori extr acts, which is likely to be the in vivo electron acceptor. Indirect ev idence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.