Investigation of the anabolic pyruvate oxidoreductase and the function of poref in methanococcus maripaludis
Lin, Winston C
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The pyruvate oxidoreductase (POR) catalyzes the reversible oxidation of pyruvate. For hydrogenotrophic methanogens, the anabolic POR catalyses the energetically unfavorable reductive carboxylation of acetyl-CoA to form pyruvate, a precursor of organic carbon synthesized in methanogens. Previously, the methanoccocal POR was purified to homogeneity. It contained five polypeptides, four of which were found to be similar to the four subunit (a, b, d, and g) POR. For further characterization, the por gene cluster was isolated and sequenced. Six open reading frames were found, porABCDEF. The gene porE encoded the fifth subunit that copurified with the POR. Also identified was porF, which was similar to porE. Both porE and porF contained high cysteinyl residue content and two Fe/S binding motifs, suggesting that porE and porF may serve as electron carriers for the methanococcal POR. To further elucidate the role of PorE and PorF, a deletional mutant strain JJ150 was constructed which lacked porEF. Growth of the mutant in minimal McN medium was slower than the growth of the wild type strain JJ1. POR, hydrogenase, and carbon monoxide dehydrogenase activities in JJ150 were similar to the wild type. In contrast, pyruvate-dependent methanogenesis was inhibited in JJ150. Complementation of the mutant with porEF restored growth and pyruvate-dependent methanogenesis to wild type levels. Partial complementation of porE or porF yielded different results. Partial complementation with porE restored methanogenesis but not growth. Complementation with porF did not restore methanogenesis but did partially restore growth. These results suggested that porEF was important for the anabolic POR. Separately, porE and porF appear to play different roles. Deletional mutagenesis of the entire por gene cluster and homologous recombination into a specific region in the por gene cluster, were performed to determine whether the POR was essential to Methanococcus. Further investigation will be needed to conclusively determine essentiality, but results suggested that the POR was essential for Methanococcus. The inactivation of porC was found to have a greater impact on the growth than the inactivation of porE. To further investigate the function of PorC, porC was deleted to form deletional mutant JJ156. However from these results, the function of porC was inconclusive.