Na+-translocating Membrane Pyrophosphatases Are Widespread in the Microbial World and Evolutionarily Precede H+-translocating Pyrophosphatases

摘要

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.