Biochemical and molecular characterization of a Na+-translocating F1Fo-ATPase from the thermoalkaliphilic bacterium Clostridium paradoxum.
Clostridium paradoxum is an anaerobic thermoalkaliphilic bacterium that grows rapidly at pH 9.8 and 56 degrees C. Under these conditions, growth is sensitive to the F-type ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), suggesting an important role for this enzyme in the physiology of C. paradoxum. The ATP synthase was characterized at the biochemical and molecular levels. The purified enzyme (30-fold purification) displayed the typical subunit pattern for an F1Fo-ATP synthase but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The purified ATPase was stimulated by sodium ions, and sodium provided protection against inhibition by DCCD that was pH dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed chemical gradient of sodium ions in the presence of a transmembrane electrical potential that was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q28, E61, and S62). On the basis of these properties, the F1Fo-ATP synthase of C. paradoxum is a sodium-translocating ATPase that is used to generate an electrochemical gradient of + that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the epsilon subunit that has been shown to be essential for coupled ATP synthesis.
Ferguson
Scott A
SA
Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin, New Zealand.
Keis
Stefanie
S
Cook
Gregory M
GM
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D027682:Cation Transport Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology*; D017124:Conserved Sequence; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013816:Thermodynamics
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D021122:Protein Subunits; D012965:Sodium Chloride; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; C021838:sodium-translocating ATPase; D006180:Proton-Translocating ATPases