title: Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases.
abstract: Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%-34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in 'canonical' mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2-PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases.
authors: *Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.
authors: Heidi H
authors: HH
authors: Luoto
authors: *Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.
authors: Erika
authors: E
authors: Nordbo
authors: *Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.
authors: Anssi M
authors: AM
authors: Malinen
authors: †Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia.
authors: Alexander A
authors: AA
authors: Baykov
authors: *Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.
authors: Reijo
authors: R
authors: Lahti
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D040143:Cellulomonas / Q000201:enzymology* / Q000235:genetics; D041883:Chlorobium / Q000201:enzymology* / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017136:Ion Transport / Q000502:physiology; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011522:Protons; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011756:Diphosphates; D008565:Membrane Proteins; D011522:Protons; D011994:Recombinant Proteins; C107241:diphosphoric acid; D012964:Sodium; D011755:Pyrophosphatases; D008274:Magnesium; D011188:Potassium