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. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.