The work described in this proposal is a continuation of our efforts to characterize membrane function and energetic in the archaebacterium Methanococcus voltae. This methanogen is a hydrogen-oxidizing obligate anaerobe which requires sodium. Studies of M. voltae membrane energetics have strongly suggested that a sodium gradient plays a central role in its energetics and that ATP synthesis may have novel features. We intend to follow up our discovery of a P-type ATPase in this methanogen by a detailed characterization of the enzyme. The enzyme will be purified, and its physical properties determined. Its ion specificity will be defined utilizing soluble and membrane-associated forms of the enzyme as well as reconstituted proteoliposomes. We will examine the ability of the enzyme to mediate the ATP-dependent generation of ion gradients and vice versa. The ATPase structural gene will be cloned and sequenced. A comparison of the derived amino acid sequence to other P-type ATPases will provide information on both the evolution and the catalytic mechanism of the enzyme. We also intend to survey closely related thermophilic methanogens for the presence of related ATPases. Comparative studies of mesophilic, moderately thermophilic, and extremely thermophilic ATPases will be performed and should lead to important insights into the structural basis for protein thermophily. We will seek to both identify membrane Na+/H+ antiporter systems and to determine the energetic basis for methyl-S-CoM uptake. Finally, we will examine possible schemes of energy transduction.
| Rusnak, P; Haney, P; Konisky, J (1995) The adenylate kinases from a mesophilic and three thermophilic methanogenic members of the Archaea. J Bacteriol 177:2977-81 |
| Chen, W; Konisky, J (1993) Characterization of a membrane-associated ATPase from Methanococcus voltae, a methanogenic member of the Archaea. J Bacteriol 175:5677-82 |
| Dybas, M; Konisky, J (1992) Energy transduction in the methanogen Methanococcus voltae is based on a sodium current. J Bacteriol 174:5575-83 |
| Dharmavaram, R; Gillevet, P; Konisky, J (1991) Nucleotide sequence of the gene encoding the vanadate-sensitive membrane-associated ATPase of Methanococcus voltae. J Bacteriol 173:2131-3 |
