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.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S.

Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics.

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed.

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.

Mechanism of Ion Translocation by Na+-Rhodopsin.

This review provides a brief description of the structure and transport function of the recently discovered family of retinal-containing Na+-translocating rhodopsins. The main emphasis is put on the kinetics of generation of electric potential difference in the membrane during a single transporter turnover. According to the proposed transport mechanism of Na+-rhodopsin, the driving force for the Na+ translocation from the cytoplasm is the local electric field created by the H+ movement from the Schiff base.

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.

A Novel, NADH-Dependent Acrylate Reductase in Vibrio harveyi.

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities.

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.

The flavin transferase ApbE flavinylates the ferredoxin:NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii.

Azotobacter vinelandii, the model microbe in nitrogen fixation studies, uses the ferredoxin:NAD+-oxidoreductase Rnf to regenerate ferredoxin (flavodoxin), acting as an electron donor for nitrogenase. However, the relative contribution of Rnf to nitrogenase functioning is unknown because this bacterium contains another ferredoxin reductase, FixABCX. Furthermore, Rnf is flavinylated in the cell, but the importance and pathway of this modification reaction also remain largely 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.

Effect of Structure Variations in the Inter-subunit Contact Zone on the Activity and Allosteric Regulation of Inorganic Pyrophosphatase from Mycobacterium tuberculosis.

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase.

A novel, cupin-type phosphoglucose isomerase in Escherichia coli.

Background: Escherichia coli cells contain a homolog of presumed 5-keto-4-deoxyuronate isomerase (KduI) from pectin-degrading soil bacteria, but the catalytic activity of the E. coli protein (o-KduI) was never demonstrated.

Methods: The known three-dimensional structure of E.

A simple strategy to differentiate between H- and Na-transporting NADH:quinone oxidoreductases.

We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na-translocating (NQR) and H-translocating (NDH-1) enzymes of the soil bacterium Azotobactervinelandii as the models. Submillimolar concentrations of Na and Li increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na.

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.

Flavodoxin with an air-stable flavin semiquinone in a green sulfur bacterium.

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld).