Membrane-bound Pyrophosphatase Research Articles

The inorganic pyrophosphatases of microorganisms: a structural and functional review

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H+/Na+ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure–function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.

Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.

Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum.

ABSTRACTThe atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk–malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h−1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate−1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive.IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk–malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.

Transcriptional activity differentiates families of Marine Group II Euryarchaeota in the coastal ocean

Marine Group II Euryarchaeota (Candidatus Poseidoniales), abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM), such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island (GA, USA) to metagenome-assembled genomes to determine the diversity of transcriptionally active Ca. Poseidoniales. Summer metatranscriptomes had the highest abundance of Ca. Poseidoniales transcripts, mostly from the O1 and O3 genera within Ca. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly from Ca. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the ß-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific to Ca. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment of Ca. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundant Ca. Poseidoniales in nearshore and inshore waters. Together, our data suggest that Ca. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.

Discovery of Membrane-Bound Pyrophosphatase Inhibitors Derived from an Isoxazole Fragment.

Membrane-bound pyrophosphatases (mPPases) regulate energy homeostasis in pathogenic protozoan parasites and lack human homologues, which makes them promising targets in e.g. malaria. Yet only few nonphosphorus inhibitors have been reported so far. Here, we explore an isoxazole fragment hit, leading to the discovery of small mPPase inhibitors with 6–10 μM IC50 values in the Thermotoga maritima test system. Promisingly, the compounds retained activity against Plasmodium falciparum mPPase in membranes and inhibited parasite growth.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor.

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids.

Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

What we have learned about membrane-bound pyrophosphatases (mPPases) from X-ray crystallography, MD simulations, FRET and PELDOR

What we have learned about membrane-bound pyrophosphatases (mPPases) from X-ray crystallography, MD simulations, FRET and PELDOR

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. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4-7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

Defining Dynamics of Membrane-Bound Pyrophosphatases by Experimental and Computational Single-Molecule FRET.

Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

A Simple Strategy to Determine the Dependence of Membrane-Bound Pyrophosphatases on K+ as a Cofactor.

Membrane-bound pyrophosphatases (mPPases) couple pyrophosphate hydrolysis to H+ and/or Na+ pumping across membranes and are found in all domains of life except for multicellular animals including humans. They are important for development and stress resistance in plants. Furthermore, mPPases play a role in virulence of human pathogens that cause severe diseases such as malaria and African sleeping sickness. Sequence analysis, functional studies, and recently solved crystal structures have contributed to the understanding of the mPPase catalytic cycle. However, several key mechanistic features remain unknown. During evolution, several subgroups of mPPases differing in their pumping specificity and cofactor dependency arose. mPPases are classified into one of five subgroups, usually by sequence analysis. However, classification based solely on sequence has been inaccurate in several instances due to our limited understanding of the molecular mechanism of mPPases. Thus, pumping specificity and cofactor dependency of mPPases require experimental confirmation. Here, we describe a simple method for the determination of K+ dependency in mPPases using a hydrolytic activity assay. By coupling these dependency studies with site-directed mutagenesis, we have begun to build a better understanding of the molecular mechanisms of mPPases. We optimized the assay for thermostable mPPases that are commonly used as model systems in our lab, but the method is equally applicable to mesophilic mPPases with minor modifications.

A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity

Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes.

Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism.

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5–6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a ‘molecular mousetrap', repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.

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. Surprisingly, we identified two mPPase lineages that independently acquired physiologically significant Na(+) and H(+) cotransport function. Na(+),H(+)-PPases of the first lineage transport H(+) over an extended [Na(+)] range, but progressively lose H(+) transport efficiency at high [Na(+)]. In contrast, H(+)-transport by Na(+),H(+)-PPases of the second lineage is not inhibited by up to 100 mM Na(+) With the identification of Na(+),H(+)-PPase subtypes, the mPPases protein superfamily appears as a continuum, ranging from monospecific Na(+) transporters to transporters with tunable levels of Na(+) and H(+) cotransport and further to monospecific H(+) transporters. Our results lend credence to the concept that Na(+) and H(+) are transported by similar mechanisms, allowing the relative efficiencies of Na(+) and H(+) transport to be modulated by minor changes in protein structure during the course of adaptation to a changing environment.

Structural basis for the reversibility of proton pyrophosphatase

ABSTRACTProton Pyrophosphatase (H+-PPase) is an evolutionarily conserved enzyme regarded as a bona fide vacuolar marker. However, H+-PPase also localizes at the plasma membrane of the phloem, where, evidence suggests that it functions as a Pyrophosphate Synthase and participates in phloem loading and photosynthate partitioning. We believe that this pyrophosphate synthesising function of H+-PPase is fundamentally rooted to its molecular structure, and here we postulate, on the basis of published crystal structures of membrane-bound pyrophosphatases, a plausible mechanism of pyrophosphate synthesis.

Membrane-bound pyrophosphatases acquired twice the physiological sodium and proton co-transport function

Membrane-bound pyrophosphatases acquired twice the physiological sodium and proton co-transport function

Membrane-bound pyrophosphatase of human gut microbe Clostridium methylpentosum confers improved salt tolerance in Escherichia coli, Saccharomyces cerevisiae and tobacco.

Membrane-bound pyrophosphatases (PPases) are involved in the adaption of organisms to stress conditions, which was substantiated by numerous plant transgenic studies with H+-PPase yet devoid of any correlated evidences for other two subfamilies, Na+-PPase and Na+,H+-PPase. Herein, we demonstrate the gene cloning and functional evaluation of the membrane-bound PPase (CmPP) of the human gut microbe Clostridium methylpentosum. The CmPP gene encodes a single polypeptide of 699 amino acids that was predicted as a multi-spanning membrane and K+-dependent Na+,H+-PPase. Heterologous expression of CmPP could significantly enhance the salt tolerance of both Escherichia coli and Saccharomyces cerevisiae, and this effect in yeast could be fortified by N-terminal addition of a vacuole-targeting signal peptide from the H+-PPase of Trypanosoma cruzi. Furthermore, introduction of CmPP could remarkably improve the salt tolerance of tobacco, implying its potential use in constructing salt-resistant transgenic crops. Consequently, the possible mechanisms of CmPP to underlie salt tolerance are discussed.

Membrane-bound pyrophosphatase: A primary proton pump

Membrane-bound pyrophosphatase: A primary proton pump

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

Membrane-bound pyrophosphatase (mPPases) of various typesconsume 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.