Rapid kinetics of H transport by membrane pyrophosphatase: Evidence for a "direct-coupling" mechanism.

Stress resistance-conferring membrane pyrophosphatase (mPPase) found in microbes and plants couples pyrophosphate hydrolysis with H transport out of the cytoplasm. There are two opposing views on the energy-coupling mechanism in this transporter: the pumping is associated with either pyrophosphate binding to mPPase or the hydrolysis step. We used our recently developed stopped-flow pyranine assay to measure H transport into mPPase-containing inverted membrane vesicles on the timescale of a single turnover.

Na Translocation Dominates over H-Translocation in the Membrane Pyrophosphatase with Dual Transport Specificity.

Cation-pumping membrane pyrophosphatases (mPPases; EC 7.1.3.

Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS Domain-Containing Pyrophosphatase.

Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,β-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction).

The Structure and Nucleotide-Binding Characteristics of Regulated Cystathionine β-Synthase Domain-Containing Pyrophosphatase without One Catalytic Domain.

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally.

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 Mechanism of Energy Coupling in H/Na-Pumping Membrane Pyrophosphatase-Possibilities and Probabilities.

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H and/or Na transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking.

A Lumenal Loop Associated with Catalytic Asymmetry in Plant Vacuolar H-Translocating Pyrophosphatase.

Membrane-integral inorganic pyrophosphatases (mPPases) couple pyrophosphate hydrolysis with H and Na pumping in plants and microbes. mPPases are homodimeric transporters with two catalytic sites facing the cytoplasm and demonstrating highly different substrate-binding affinities and activities. The structural aspects of the functional asymmetry are still poorly understood because the structure of the physiologically relevant dimer form with only one active site occupied by the substrate is unknown.

Catalytic Asymmetry in Homodimeric H-Pumping Membrane Pyrophosphatase Demonstrated by Non-Hydrolyzable Pyrophosphate Analogs.

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H and Na transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a "direct coupling" mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood.

Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities.

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation.

Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli.

Background: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA).

Methods: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure.

Results: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI.

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.

Mutational analysis of the flavinylation and binding motifs in two protein targets of the flavin transferase ApbE.

Many flavoproteins belonging to three domain types contain an FMN residue linked through a phosphoester bond to a threonine or serine residue found in a conserved seven-residue motif. The flavinylation reaction is catalyzed by a specific enzyme, ApbE, which uses FAD as a substrate. To determine the structural requirements of the flavinylation reaction, we examined the effects of single substitutions in the flavinylation motif of Klebsiella pneumoniae cytoplasmic fumarate reductase on its modification by its own ApbE in recombinant Escherichia coli cells.

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.

Roles of nucleotide substructures in the regulation of cystathionine β-synthase domain-containing pyrophosphatase.

Background: Regulatory cystathionine β-synthase (CBS) domains are ubiquitous in proteins, yet their mechanism of regulation remains largely obscure. Inorganic pyrophosphatase which contains regulatory CBS domains as internal inhibitors (CBS-PPase) is activated by ATP and inhibited by AMP and ADP; nucleotide binding to CBS domains and substrate binding to catalytic domains demonstrate positive co-operativity.

Methods: Here, we explore the ability of an AMP analogue (cAMP) and four compounds that mimic the constituent parts of the AMP molecule (adenine, adenosine, phosphate, and fructose-1-phosphate) to bind and alter the activity of CBS-PPase from the bacterium Desulfitobacterium hafniense.

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.

Identification of the key determinant of the transport promiscuity in Na-translocating rhodopsins.

Bacterial Na-transporting rhodopsins convert solar energy into transmembrane ion potential difference. Typically, they are strictly specific for Na, but some can additionally transport H. To determine the structural basis of cation promiscuity in Na-rhodopsins, we compared their primary structures and found a single position that harbors a cysteine in strictly specific Na-rhodopsins and a serine in the promiscuous Krokinobacter eikastus Na-rhodopsin (Kr2).

Rat ceruloplasmin: a new labile copper binding site and zinc/copper mosaic.

Ceruloplasmin (Cp) is a copper-containing multifunctional oxidase of plasma, an antioxidant, an acute-phase protein and a free radical scavenger. The structural organization of Cp causes its sensitivity to proteolysis and ROS (reactive oxygen species), which can alter some of the important Cp functions. Elucidation of the orthorhombic crystal structure of rat Cp at 2.

Enzymes Regulated via Cystathionine β-Synthase Domains.

Cystathionine β-synthase (CBS) domains discovered 20 years ago can bind different adenosine derivatives (AMP, ADP, ATP, S-adenosylmethionine, NAD, diadenosine polyphosphates) and thus regulate the activities of numerous proteins. Mutations in CBS domains of enzymes and membrane transporters are associated with several hereditary diseases. The regulatory unit is a quartet of CBS domains that belong to one or two polypeptides and usually form a conserved disk-like structure.

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.

Engineering a carotenoid-binding site in Dokdonia sp. PRO95 Na-translocating rhodopsin by a single amino acid substitution.

Light-driven H, Cl and Na rhodopsin pumps all use a covalently bound retinal molecule to capture light energy. Some H-pumping rhodopsins (xanthorhodopsins; XRs) additionally contain a carotenoid antenna for light absorption. Comparison of the available primary and tertiary structures of rhodopsins pinpointed a single Thr residue (Thr216) that presumably prevents carotenoid binding to Na-pumping rhodopsins (NaRs).

Inhibition of Escherichia coli Inorganic Pyrophosphatase by Fructose-1-phosphate.

Pyrophosphate regulates vital cellular reactions, and its level in E. coli cells is under the ultimate control of inorganic pyrophosphatase. The mechanisms involved in the regulation of pyrophosphatase activity still need to be elucidated.

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).