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

Abstract

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.

Membrane-integral pyrophosphatase subfamily capable of translocating both Na + and H +

“…In the presence of 50 mM K + , the curve shifted markedly to lower Na + concentrations, and maximal achievable activity increased. The rate data were quantitatively modeled in terms of Scheme 1, which assumes the binding of one activating and one inhibitory Na + ion to Bv-PPase, similar to previous studies of Na + -PPases (8). The best-fit parameter values, summarized in Table 1, indicate that at pH 7.2, K + induces a 60-fold decrease in the dissociation constant for the activating Na + ion (K a ), no change in inhibitory Na + ion binding (K i ), and a 1.2-fold increase in the activity of the monosodium complex (V 1 ).…”

“…Replacement of the Na + -PPase Asp-Lys-Glu channel gate (6) with Asp-Lys is possibly the key event in conferring H + selectivity to plant-type PPases (5), despite the close similarities of the ion conductance channels. Other structural solutions to control the identity of transported ions have additionally been used in the evolutionary history of the PPase superfamily, which includes several independent transitions from Na + to H + transport (8). All previously characterized membrane PPases have been shown to operate as either specific H + or Na + transporters.…”

“…All membrane PPases requiring Na + for activity operate as primary Na + transporters (1,8,9). Accordingly, addition of PP i to the reaction medium caused accumulation of 22 …”

“…To estimate the proportion of membrane PPases in which Na + and H + transport are intrinsically coupled, we constructed a Bayesian phylogeny of the protein superfamily using a previously described approach (8). The sequence set was supplemented with Bv-PPase, and a few similar sequences identified in the National Center for Biotechnology Information (NCBI) nonredundant protein sequence (nr) databank (10).…”

Structural basis for the reversibility of proton pyrophosphatase

“…2), a residue, which according to site-directed mutagenesis data, is indispensable for ion translocation. [31][32][33][34] The strategic positive charge on the E301 residue facilitates PPi synthesis by stabilizing at the transition state conformation, the negatively charged hydroxyl ion released: Pi 2¡ C Pi 2¡ ! PPi 3¡ C OH ¡ (Step 2; Fig.…”