Pre-steady-state kinetics and solvent isotope effects support the "billiard-type" transport mechanism in Na -translocating pyrophosphatase.

Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H pump that transports the H ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism.

The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding.

A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200-250-kDa homotetramers, which seems unprecedented among the four PPase families.

Residue Network Involved in the Allosteric Regulation of Cystathionine β-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides.

Inorganic pyrophosphatase containing regulatory cystathionine β-synthase (CBS) domains (CBS-PPase) is inhibited by adenosine monophosphate (AMP) and adenosine diphosphate and activated by adenosine triphosphate (ATP) and diadenosine polyphosphates; mononucleotide binding to CBS domains and substrate binding to catalytic domains are characterized by positive cooperativity. This behavior implies three pathways for regulatory signal transduction - between regulatory and active sites, between two active sites, and between two regulatory sites. Bioinformatics analysis pinpointed six charged or polar amino acid residues of CBS-PPase as potentially important for enzyme regulation.

Cooperativity in catalysis by canonical family II pyrophosphatases.

Bacterial family II pyrophosphatases (PPases) are homodimeric enzymes, with the active site located between two catalytic domains. Some family II PPases additionally contain regulatory cystathionine β-synthase (CBS) domains and exhibit positive kinetic cooperativity, which is lost upon CBS domain removal. We report here that CBS domain-deficient family II PPases of Bacillus subtilis and Streptococcus gordonii also exhibit positive kinetic cooperativity, manifested as an up to a five-fold difference in the Michaelis constants for two active sites.

An arginine residue involved in allosteric regulation of cystathionine β-synthase (CBS) domain-containing pyrophosphatase.

Inorganic pyrophosphatase containing a pair of regulatory CBS domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and diadenosine polyphosphates. Mononucleotide binding to CBS domains and substrate binding to catalytic domains are characterized by positive co-operativity. Bioinformatics analysis pinpointed a conserved arginine residue at the interface of the regulatory and catalytic domains in bacterial CBS-PPases as potentially involved in enzyme regulation.

Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases.

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H and/or Na ions, are divided into K,Na-independent, Na-regulated, and K-dependent families. The first two families include H-transporting mPPases (H-PPases), whereas the last family comprises one Na-transporting, two Na- and H-transporting subfamilies (Na-PPases and Na,H-PPases, respectively), and three H-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K-independent mPPases.

Inorganic pyrophosphatases of Family II-two decades after their discovery.

Inorganic pyrophosphatases (PPases) convert pyrophosphate (PP ) to phosphate and are present in all cell types. Soluble PPases belong to three nonhomologous families, of which Family II is found in approximately a quarter of prokaryotic organisms, often pathogenic ones. Each subunit of dimeric canonical Family II PPases is formed by two domains connected by a flexible linker, with the active site located between the domains.

Two independent evolutionary routes to Na+/H+ cotransport function in membrane pyrophosphatases.

Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi) to transport H(+), Na(+) or both and help organisms to cope with stress conditions, such as high salinity or limiting nutrients. Recent elucidation of mPPase structure and identification of subfamilies that have fully or partially switched from Na(+) to H(+) pumping have established mPPases as versatile models for studying the principles governing the mechanism, specificity and evolution of cation transporters. In the present study, we constructed an accurate phylogenetic map of the interface of Na(+)-transporting PPases (Na(+)-PPases) and Na(+)- and H(+)-transporting PPases (Na(+),H(+)-PPases), which guided our experimental exploration of the variations in PPi hydrolysis and ion transport activities during evolution.

An asparagine residue mediates intramolecular communication in nucleotide-regulated pyrophosphatase.

Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS (cystathionine β-synthase) domains that act as 'internal inhibitors' whose effect is modulated by nucleotide binding. Although such regulatory domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS domain-containing PPases).

Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria.

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively.

Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase.

Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood.

Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases.

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).

Cystathionine β-synthase (CBS) domains confer multiple forms of Mg2+-dependent cooperativity to family II pyrophosphatases.

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine β-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta.

Membrane Na+-pyrophosphatases can transport protons at low sodium concentrations.

Membrane-bound Na(+)-pyrophosphatase (Na(+)-PPase), working in parallel with the corresponding ATP-energized pumps, catalyzes active Na(+) transport in bacteria and archaea. Each ~75-kDa subunit of homodimeric Na(+)-PPase forms an unusual funnel-like structure with a catalytic site in the cytoplasmic part and a hydrophilic gated channel in the membrane. Here, we show that at subphysiological Na(+) concentrations (<5 mM), the Na(+)-PPases of Chlorobium limicola, four other bacteria, and one archaeon additionally exhibit an H(+)-pumping activity in inverted membrane vesicles prepared from recombinant Escherichia coli strains.

Pyrophosphate-fueled Na+ and H+ transport in prokaryotes.

In its early history, life appeared to depend on pyrophosphate rather than ATP as the source of energy. Ancient membrane pyrophosphatases that couple pyrophosphate hydrolysis to active H(+) transport across biological membranes (H(+)-pyrophosphatases) have long been known in prokaryotes, plants, and protists. Recent studies have identified two evolutionarily related and widespread prokaryotic relics that can pump Na(+) (Na(+)-pyrophosphatase) or both Na(+) and H(+) (Na(+),H(+)-pyrophosphatase).

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

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H(+)-transporting (found in prokaryotes, protists, and plants) and Na(+)-transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport.

The CBS domain: a protein module with an emerging prominent role in regulation.

Regulatory CBS (cystathionine β-synthase) domains exist as two or four tandem copies in thousands of cytosolic and membrane-associated proteins from all kingdoms of life. Mutations in the CBS domains of human enzymes and membrane channels are associated with an array of hereditary diseases. Four CBS domains encoded within a single polypeptide or two identical polypeptides (each having a pair of CBS domains at the subunit interface) form a highly conserved disk-like structure.

Na+-translocating membrane pyrophosphatases are widespread in the microbial world and evolutionarily precede H+-translocating pyrophosphatases.

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.

Mutational analysis of residues in the regulatory CBS domains of Moorella thermoacetica pyrophosphatase corresponding to disease-related residues of human proteins.

mtCBS-PPase [CBS (cystathionine β-synthase) domain-containing pyrophosphatase from Moorella thermoacetica] contains a pair of CBS domains that strongly bind adenine nucleotides, thereby regulating enzyme activity. Eight residues associated with the CBS domains of mtCBS-PPase were screened to explore possible associations with regulation of enzyme activity. The majority of the substitutions (V99A, R168A, Y169A, Y169F, Y188A and H189A) enhanced the catalytic activity of mtCBS-PPase, two substitutions (R170A and R187G) decreased activity, and one substitution (K100G) had no effect.

Nucleotide- and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica.

In contrast to all other known pyrophosphatases, Moorella thermoacetica pyrophosphatase (mtCBS-PPase) contains nucleotide-binding CBS domains and is thus strongly regulated by adenine nucleotides. Stopped-flow measurements using a fluorescent AMP analogue, 2'(3')-O-(N-methylanthranoyl)-AMP (Mant-AMP), reveal that nucleotide binding to mtCBS-PPase involves a three-step increase in Mant-AMP fluorescence with relaxation times from 0.01 to 100 s, implying conformational changes in the complex.

Mutual effects of cationic ligands and substrate on activity of the Na+-transporting pyrophosphatase of Methanosarcina mazei.

The PP(i)-driven sodium pump (membrane pyrophosphatase) of Methanosarcina mazei (Mm-PPase) absolutely requires Na(+) and Mg(2+) for activity and additionally employs K(+) as a modulating cation. Here we explore relationships among Na(+), K(+), Mg(2+), and PP(i) binding sites by analyzing the dependency of the Mm-PPase PP(i)-hydrolyzing function on these ligands and protection offered by the ligands against Mm-PPase inactivation by trypsin and the SH-reagent mersalyl. Steady-state kinetic analysis of PP(i) hydrolysis indicated that catalysis involves random order binding of two Mg(2+) ions and two Na(+) ions, and the binding was almost independent of substrate (Mg(2)PP(i) complex) attachment.

Human metastasis regulator protein H-prune is a short-chain exopolyphosphatase.

The DHH superfamily human protein h-prune, a binding partner of the metastasis suppressor nm23-H1, is frequently overexpressed in metastatic cancers. From an evolutionary perspective, h-prune is very close to eukaryotic exopolyphosphatases. Here, we show for the first time that h-prune efficiently hydrolyzes short-chain polyphosphates (k cat of 3-40 s (-1)), including inorganic tripoly- and tetrapolyphosphates and nucleoside 5'-tetraphosphates.

A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides.

CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains.

Na+-pyrophosphatase: a novel primary sodium pump.

Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from the hyperthermophilic bacterium Thermotoga maritima catalyze Na+ rather than H+ transport into Escherichia coli inner membrane vesicles (IMV). When assayed in uncoupled IMV, the three PPases exhibit an absolute requirement for Na+ but display the highest hydrolyzing activity in the presence of both Na+ and K+.

A complete structural description of the catalytic cycle of yeast pyrophosphatase.

We have determined the structures of the wild type and seven active site variants of yeast inorganic pyrophosphatase (PPase) in the presence of Mg2+ and phosphate, providing the first complete structural description of its catalytic cycle. PPases catalyze the hydrolysis of pyrophosphate and require four divalent metal cations for catalysis; magnesium provides the highest activity. The crystal form chosen contains two monomers in the asymmetric unit, corresponding to distinct catalytic intermediates.