title: Na+-translocating membrane pyrophosphatases are widespread in the microbial world and evolutionarily precede H+-translocating pyrophosphatases.
abstract: Membrane pyrophosphatases (PPases), divided into K(+)-dependent and K(+)-independent subfamilies, were believed to pump H(+) across cell membranes until a recent demonstration that some K(+)-dependent PPases function as Na(+) pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na(+) transport, and H(+) transport activities as well as their K(+) and Na(+) requirements in inner membrane vesicles. Four of these enzymes (from Anaerostipes caccae, Chlorobium limicola, Clostridium tetani, and Desulfuromonas acetoxidans) were identified as K(+)-dependent Na(+) transporters. Phylogenetic analysis led to the identification of a monophyletic clade comprising characterized and predicted Na(+)-transporting PPases (Na(+)-PPases) within the K(+)-dependent subfamily. H(+)-transporting PPases (H(+)-PPases) are more heterogeneous and form at least three independent clades in both subfamilies. These results suggest that rather than being a curious rarity, Na(+)-PPases predominantly constitute the K(+)-dependent subfamily. Furthermore, Na(+)-PPases possibly preceded H(+)-PPases in evolution, and transition from Na(+) to H(+) transport may have occurred in several independent enzyme lineages. Site-directed mutagenesis studies facilitated the identification of a specific Glu residue that appears to be central in the transport mechanism. This residue is located in the cytoplasm-membrane interface of transmembrane helix 6 in Na(+)-PPases but shifted to within the membrane or helix 5 in H(+)-PPases. These results contribute to the prediction of the transport specificity and K(+) dependence for a particular membrane PPase sequence based on its position in the phylogenetic tree, identity of residues in the K(+) dependence signature, and position of the membrane-located Glu residue.
authors.affiliation: Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
authors.forename: Heidi H
authors.initials: HH
authors.lastname: Luoto
authors.forename: Georgiy A
authors.initials: GA
authors.lastname: Belogurov
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Baykov
authors.forename: Reijo
authors.initials: R
authors.lastname: Lahti
authors.forename: Anssi M
authors.initials: AM
authors.lastname: Malinen
mesh_terms: D001692:Biological Transport; D002412:Cations; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D019143:Evolution, Molecular; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D011188:Potassium / Q000737:chemistry; D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D012964:Sodium / Q000737:chemistry / Q000378:metabolism
chemical_list: D002412:Cations; D008024:Ligands; D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins; D012964:Sodium; D043564:Inorganic Pyrophosphatase; D011188:Potassium