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 PP(i)-hydrolyzing and PP(i)-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. hydrogenoformans H(+)-PPase is sufficient to confer K(+) independence to both PP(i) hydrolysis and PP(i)-energized H(+) translocation. In contrast, a A463T mutation does not affect the K(+) dependence of H(+)-PPase.
Belogurov
Georgiy A
GA
Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland. geobel@utu.fi
Lahti
Reijo
R
D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D008239:Lysine / Q000737:chemistry*; D009154:Mutation; D010459:Peptococcaceae / Q000201:enzymology*; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry* / Q000494:pharmacology; D021381:Protein Transport; D011522:Protons; D011755:Pyrophosphatases / Q000235:genetics* / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry
D011522:Protons; D013912:Threonine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008239:Lysine; D011188:Potassium