Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori

Abstract

CO 3؊ 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.Helicobacter pylori is a microaerophilic, gram-negative, motile, curved or spiral bacterium, isolated from the mucous layer overlying the human gastric epithelium (35,53). To date, eleven Helicobacter species have been isolated from a range of mammals (15,20,32). The organism is now accepted as the etiological agent for type B gastritis, and extensive evidence suggests a link with duodenal and gastric ulcer diseases (17,21,25,47,52). Individuals with long-term infections may also be at increased risk of developing gastric carcinoma (44).Enzyme activities identified in this bacterium include catalase, cytochrome oxidase, superoxide dismutase, and phospholipase, and it is a constitutive producer of a potent urease (18,23). Pathogenicity has been associated with adhesion to the gastric epithelium (7,24,43), urease production (36), a vacuolating cytotoxin (10,11,13), and the action of phospholipases (31). However, knowledge of the basic metabolism of H. pylori is meager. This organism exhibits limited glucose utilization (39, 41), but it is reported to contain pentose phosphate (38) and Entner-Douderoff pathways (8,42). Recently, utilization of glucose as a carbon and energy source in a defined medium has been shown (48). Active fumarate catabolism, generating malate and succinate from fumarate initially, followed by the formation of lactate, acetate, formate, and alanine, has been demonstrated (40). Suspensions of intact cells have been shown to generate lactate, ethanol, alanine, acetate, and CO 2 from pyruvate (8). This organism also requires elevated levels of CO 2 for growth, and it has been suggested that potent urease activity in part feeds this requirement (22). HCO 3 Ϫ is used in de novo synthesis of pyrimidines, but the demand for CO 2 considerably exceeds that of HCO 3 Ϫ . Gaseous CO 2 cannot be replaced by the addition of bicarbonate to the medium (22).Microorganisms exhibiting dependence on elevated CO 2 levels are termed capneic or capnophilic. Perhaps the best examples of pathogenic capnophiles are Neisseria spp. The enzymes known to be involved in CO 2 assimilation by Neisseria spp. are carbonic anhydrase (34) and phosphoenolpyruvate (PEP) carboxylase (12). Pyruvate carboxylase, PEP carboxykinase, and PEP carboxytransphosphorylase have not been dete...

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

“…Another possibility would be that these POR might be differentially expressed at different growth temperatures. While the 4 subunit type of POR seems to be most common in hyperthermophiles [33], its presence has also been observed in mesophiles [34]. Conversely the single subunit type, also found in the mesophile C. acetobutylicum has an in vitro temperature optimum of 60°C [21].…”

Enhanced rat β-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway

“…The fold increase in PC activity is the same as what we found by the previous method, but the absolute values are lower than our previous results (26). The method of determining PC activity by measuring NADH production has been widely used by many authors for determination of PC (10,15,50,58) and LDH activity (1,14,17,40,60) (only a partial list) in many different tissues. Because this method cannot discriminate between PC and LDH activities, the results reflect the total activity of PC plus LDH, which can be misleading when interpreted as only PC or LDH.…”

A Complete Sequence of the T. tengcongensis Genome

“…Similar to the cases of Helicobacter pylori and Campylobacter jejuni , T. tengcongensis has 12 genes (TTE0445, TTE0960, TTE0961, TTE1209, TTE1210, TTE1211, TTE1340, TTE1341, TTE1342, TTE2193, TTE2194, and TTE2198) related to the pyruvate:ferredoxin oxidoreductases and 2-oxoacid:ferredoxin oxidoreductases. The conversion of pyruvate to acetyl coenzyme A (acetyl CoA) is performed by the pyruvate ferrodoxin oxidoreductase (POR; Cayol et al 1995;Menon and Ragsdale 1997), a foursubunit enzyme described in H. pylori and other hyperthermophilic organisms (Hughes et al 1995). Acetyl CoA is converted to acetate and this process is catalyzed by four enzymes, phosphate acetyltransferase (TTE1482, TTE2195, and TTE2204), acetate kinase (TTE1481), NADH-:flavin oxidoreductase (TTE0012, TTE0988, TTE2131, and TTE2625), and Acyl-CoA dehydrogenase (TTE0545; Bock et al 1999).…”