A lysine substitute for K+ -: A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans

The H+ proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-PPase from the thermophilic bacterium Carboxydothermus hydrogenoformans. Both PPi-hydrolyzing and PPi-energized H+ translocation activities of the recombinant enzyme in Escherichia coli inner membrane vesicles are strictly K+-dependent. Here we deduce the K+ requirement of all available H+-PPase sequences based on the K+ dependence of C hydrogenoformans H+-PPase in conjunction with phylogenetic analyses. Our data reveal that K+-independent H+-PPases possess conserved Lys and Thr that are absent in K+-dependent H+-PPases. We further demonstrate that a A460K substitution in C. hydrog-enoformans H+-PPase is sufficient to confer K+ independence to both PPi hydrolysis and PPi-energized H+ translocation. In contrast, a A463T mutation does not affect the K+ dependence of H+-PPase.