Publications by authors named "Alexander A Baykov"

53 Publications

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

Molecules 2021 Apr 18;26(8). Epub 2021 Apr 18.

Department of Life Technologies, University of Turku, FIN-20014 Turku, Finland.

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. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP hydrolysis, (b) continuous and fixed-time measurements of PP synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.
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http://dx.doi.org/10.3390/molecules26082356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073611PMC
April 2021

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

Biochim Biophys Acta Gen Subj 2021 01 11;1865(1):129762. Epub 2020 Oct 11.

Department of Chemistry and Belozersky, Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russian Federation. Electronic address:

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. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers.

Conclusions: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis.

General Significance: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.
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http://dx.doi.org/10.1016/j.bbagen.2020.129762DOI Listing
January 2021

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

Arch Biochem Biophys 2020 10 15;692:108537. Epub 2020 Aug 15.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia. Electronic address:

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. The tetrameric structure is stabilized by Co, the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation.
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http://dx.doi.org/10.1016/j.abb.2020.108537DOI Listing
October 2020

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

Biochim Biophys Acta Gen Subj 2020 07 13;1864(7):129601. Epub 2020 Mar 13.

Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia. Electronic address:

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. coli o-KduI was compared with the available structures of sugar-converting enzymes. Based on the results of this analysis, sugar isomerization activity of recombinant o-KduI was tested against a panel of D-sugars and their derivatives.

Results: The three-dimensional structure of o-KduI exhibits a close similarity with Pyrococcus furiosus cupin-type phosphoglucose isomerase. In accordance with this similarity, o-KduI was found to catalyze interconversion of glucose-6-phosphate and fructose-6-phosphate and, less efficiently, conversion of glucuronate to fructuronate. o-KduI was hexameric in crystals but represented a mixture of inactive hexamers and active dimers in solution and contained a tightly bound Zn ion. Dilution, substrate binding and Zn removal shifted the hexamer ⇆ dimer equilibrium to the dimers.

Conclusions: Our findings identify o-KduI as a novel phosphosugar isomerase in E. coli, whose activity may be regulated by changes in oligomeric structure.

General Significance: More than 5700 protein sequences are annotated as KduI, but their enzymatic activity has not been directly demonstrated. E. coli o-KduI is the first characterized member of this group, and its enzymatic activity was found to be different from the predicted activity.
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http://dx.doi.org/10.1016/j.bbagen.2020.129601DOI Listing
July 2020

Energy Coupling in Cation-Pumping Pyrophosphatase-Back to Mitchell.

Front Plant Sci 2020 14;11:107. Epub 2020 Feb 14.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

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http://dx.doi.org/10.3389/fpls.2020.00107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034417PMC
February 2020

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

Arch Biochem Biophys 2020 03 15;681:108266. Epub 2020 Jan 15.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119234, Russia. Electronic address:

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. These findings testified a primary Na-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na or H transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na transport in nitrogen fixation by A. vinelandii.
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http://dx.doi.org/10.1016/j.abb.2020.108266DOI Listing
March 2020

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

FEMS Microbiol Lett 2019 11;366(22)

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Vorobievy Gory 1/40, Moscow 119234, Russia.

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. The replacement of the flavin acceptor threonine with alanine completely abolished the modification reaction, whereas the replacements of conserved aspartate and serine had only minor effects. Effects of other substitutions, including replacing the acceptor threonine with serine, (a 10-55% decrease in the flavinylation degree) pinpointed important glycine and alanine residues and suggested an excessive capacity of the ApbE-based flavinylation system in vivo. Consistent with this deduction, drastic replacements of conserved leucine and threonine residues in the binding pocket that accommodates FMN residue still allowed appreciable flavinylation of the NqrC subunit of Vibrio harveyi Na+-translocating NADH:quinone oxidoreductase, despite a profound weakening of the isoalloxazine ring binding and an increase in its exposure to solvent.
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http://dx.doi.org/10.1093/femsle/fnz252DOI Listing
November 2019

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

ACS Omega 2019 Sep 10;4(13):15549-15559. Epub 2019 Sep 10.

Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia.

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. Twelve mutant enzyme forms were produced, and their kinetics of pyrophosphate hydrolysis was measured in wide concentration ranges of the substrate and various adenine nucleotides. The parameters derived from this analysis included catalytic activity, Michaelis constants for two active sites, AMP-, ATP-, and diadenosine tetraphosphate-binding constants for two regulatory sites, and the degree of activation/inhibition for each nucleotide. Replacements of arginine 295 and asparagine 312 by alanine converted ATP from an activator to an inhibitor and markedly affected practically all the above parameters, indicating involvement of these residues in all the three regulatory signaling pathways. Replacements of asparagine 312 and arginine 334 abolished or reversed kinetic cooperativity in the absence of nucleotides but conferred it in the presence of diadenosine tetraphosphate, without effects on nucleotide-binding parameters. Modeling and molecular dynamics simulations revealed destabilization of the subunit interface as a result of asparagine 312 and arginine 334 replacements by alanine, explaining abolishment of kinetic cooperativity. These findings identify residues 295, 312, and 334 as crucial for CBS-PPase regulation via CBS domains.
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http://dx.doi.org/10.1021/acsomega.9b01879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761619PMC
September 2019

Cooperativity in catalysis by canonical family II pyrophosphatases.

Biochem Biophys Res Commun 2019 09 23;517(2):266-271. Epub 2019 Jul 23.

Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia. Electronic address:

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 Asn79Ser replacement in S. gordonii PPase preserved its dimeric structure but abolished cooperativity. The results of our study indicated that kinetic cooperativity is an inherent property of all family II PPase types, is not induced by CBS domains, and is sensitive to minor structural changes. These findings may have inferences for other CBS-proteins, which include important enzymes and membrane transporters associated with hereditary diseases.
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http://dx.doi.org/10.1016/j.bbrc.2019.07.056DOI Listing
September 2019

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

Photosynth Res 2019 Nov 13;142(2):127-136. Epub 2019 Jul 13.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 119234.

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). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.
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http://dx.doi.org/10.1007/s11120-019-00658-1DOI Listing
November 2019

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

Biochim Biophys Acta Gen Subj 2019 08 17;1863(8):1263-1269. Epub 2019 May 17.

Belozersky Institute of Physico-Chemical Biology, Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russian Federation. Electronic address:

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.

Results: Adenine, adenosine and cAMP activated CBS-PPase several-fold whereas fructose-1-phosphate inhibited it. Adenine and adenosine binding to dimeric CBS-PPase exhibited high positive co-operativity and markedly increased substrate binding co-operativity. Phosphate bound to CBS-PPase competitively with respect to a fluorescent AMP analogue.

Conclusions: Protein interactions with the adenine moiety of AMP induce partial release of the internal inhibition and determine nucleotide-binding co-operativity, whereas interactions with the phosphate group potentiate the internal inhibition and decrease active-site co-operativity. The ribose moiety appears to enhance the activation effect of adenine and suppress its contribution to both types of co-operativity.

General Significance: Our findings demonstrate for the first time that regulation of a CBS-protein (inhibition or activation) is determined by a balance of its interactions with different chemical groups of the nucleotide and can be reversed by their modification. Differential regulation by nucleotides is not uncommon among CBS-proteins, and our findings may thus have a wider significance.
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http://dx.doi.org/10.1016/j.bbagen.2019.05.010DOI Listing
August 2019

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

Arch Biochem Biophys 2019 02 28;662:40-48. Epub 2018 Nov 28.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899, Russia. Electronic address:

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. The importance of this residue was assessed by site-directed mutagenesis using the CBS-PPase from Desulfitobacterium hafniense (dhPPase) as a model. The mutants R276A, R276K and R276E were constructed and purified, and the impact of the respective mutation on catalysis, nucleotide binding and regulation was analysed. Overall, the effects decreased in the following order R276A > R276E > R276K. The variants retained ≥50% catalytic efficiency but exhibited reduced kinetic co-operativity or even its inversion (R276A). Negative co-operativity was retained in the R276A variant in the presence of mononucleotides but was reversed by diadenosine tetraphosphate. Positive nucleotide-binding co-operativity was retained in all variants but the R276A and R276E variants exhibited a markedly reduced affinity to AMP and ADP and greater residual activity at their saturating concentrations. The R276A substitution abolished activation by ATP and diadenosine tetraphosphate, while preserving the ability to bind them. The results suggest that the H-bond formed by the Arg276 sidechain is essential for signal transduction between the regulatory and catalytic domains within one subunit and between the catalytic but not regulatory domains of different subunits.
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http://dx.doi.org/10.1016/j.abb.2018.11.024DOI Listing
February 2019

Flavin transferase: the maturation factor of flavin-containing oxidoreductases.

Biochem Soc Trans 2018 10 28;46(5):1161-1169. Epub 2018 Aug 28.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia.

Flavins, cofactors of many enzymes, are often covalently linked to these enzymes; for instance, flavin adenine mononucleotide (FMN) can form a covalent bond through either its phosphate or isoalloxazine group. The prevailing view had long been that all types of covalent attachment of flavins occur as autocatalytic reactions; however, in 2013, the first flavin transferase was identified, which catalyzes phosphoester bond formation between FMN and Na-translocating NADH:quinone oxidoreductase in certain bacteria. Later studies have indicated that this post-translational modification is widespread in prokaryotes and is even found in some eukaryotes. Flavin transferase can occur as a separate ∼40 kDa protein or as a domain within the target protein and recognizes a degenerate DgxtsA/ motif in various target proteins. The purpose of this review was to summarize the progress already achieved by studies of the structure, mechanism, and specificity of flavin transferase and to encourage future research on this topic. Interestingly, the flavin transferase gene (E) is found in many bacteria that have no known target protein, suggesting the presence of yet unknown flavinylation targets.
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http://dx.doi.org/10.1042/BST20180524DOI Listing
October 2018

Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase.

Biochimie 2018 Jun 3;149:34-40. Epub 2018 Apr 3.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia. Electronic address:

One of the three domains of kinetoplastid NADH:fumarate oxidoreductase (FRD) is homologous to bacterial flavin transferase that catalyzes transfer of FMN residue from FAD to threonine in flavoproteins. Leptomonas pyrrhocoris FRD produced in yeast cells, which lack flavin transferase gene in their proteome, reduces fumarate in the presence of NADH and contains an FMN residue covalently linked to a Ser9 residue. The conserved flavinylation motif of FRD, D(g/s)x(s/t)(s/g)AS, is similar to the Dxx(s/t)gAT motif recognized by flavin transferase in prokaryotic proteins. Ser9 replacement abolished the flavinylation and fumarate reductase activity of FRD. These findings suggest that the flavinylation is important for the activity of FRD and that this post-translational modification is carried out by the own flavin transferase domain.
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http://dx.doi.org/10.1016/j.biochi.2018.03.013DOI Listing
June 2018

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

Biochem Biophys Res Commun 2018 05 30;499(3):600-604. Epub 2018 Mar 30.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia. Electronic address:

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). A Cys253Ser variant of the strictly specific Dokdonia sp. PRO95 Na-rhodopsin (NaR) was indeed found to transport both Na and H in a light-dependent manner when expressed in retinal-producing Escherichia coli cells. The dual specificity of the NaR variant was confirmed by analysis of its photocycle, which revealed an acceleration of the cation-capture step by comparison with the wild-type NaR in a Na-deficient medium. The structural basis for the dependence of the Na/H specificity in Na-rhodopsin on residue 253 remains to be determined.
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http://dx.doi.org/10.1016/j.bbrc.2018.03.196DOI Listing
May 2018

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

Biochem J 2018 03 26;475(6):1141-1158. Epub 2018 Mar 26.

Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland

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.
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http://dx.doi.org/10.1042/BCJ20180071DOI Listing
March 2018

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

FEBS Lett 2017 10 17;591(20):3225-3234. Epub 2017 Oct 17.

Department of Biochemistry, University of Turku, Finland.

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. These enzymes require both magnesium and a transition metal ion (manganese or cobalt) for maximal activity and are the most active (k ≈ 10 s ) among all PPase types. Catalysis by Family II PPases requires four metal ions per substrate molecule, three of which form a unique trimetal center that coordinates the nucleophilic water and converts it to a reactive hydroxide ion. A quarter of Family II PPases contain an autoinhibitory regulatory insert formed by two cystathionine β-synthase (CBS) domains and one DRTGG domain. Adenine nucleotide binding either activates or inhibits the CBS domain-containing PPases, thereby tuning their activity and, hence, PP levels, in response to changes in cell energy status (ATP/ADP ratio).
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http://dx.doi.org/10.1002/1873-3468.12877DOI Listing
October 2017

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

Photosynth Res 2018 May 5;136(2):161-169. Epub 2017 Oct 5.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia, 119234.

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). We replaced this residue in Dokdonia sp. PRO95 NaR with Gly, which is found in the corresponding position in XRs, and produced a variant rhodopsin in a ketocarotenoid-synthesising Escherichia coli strain. Unlike wild-type NaR, the isolated variant protein contained the tightly bound carotenoids canthaxanthin and echinenone. These carotenoids were visible in the absorption, circular dichroism and fluorescence excitation spectra of the Thr216Gly-substituted NaR, which indicates their function as a light-harvesting antenna. The amino acid substitution and the bound carotenoids did not affect the NaR photocycle. Our findings suggest that the antenna function was recently lost during NaR evolution but can be easily restored by site-directed mutagenesis.
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http://dx.doi.org/10.1007/s11120-017-0453-0DOI Listing
May 2018

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

Biochem J 2016 10 3;473(19):3099-111. Epub 2016 Aug 3.

Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland.

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.
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http://dx.doi.org/10.1042/BCJ20160529DOI Listing
October 2016

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

Biochem J 2016 07 17;473(14):2097-107. Epub 2016 May 17.

Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia

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). In the present study, we demonstrate that a homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an inherent mutation in an otherwise conserved asparagine residue in a loop near the active site exhibits non-co-operative hydrolysis kinetics. A similar N312S substitution in 'co-operative' dhPPase (Desulfitobacterium hafniense PPase) abolished kinetic co-operativity while causing only minor effects on nucleotide-binding affinity and co-operativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase, but restoring it in the variant dhPPase. A reverse serine-to-asparagine replacement restored kinetic co-operativity in ehPPase. Molecular dynamics simulations revealed that the asparagine substitution resulted in a change in the hydrogen-bonding pattern around the asparagine residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this asparagine residue as lying at the 'crossroads' of information paths connecting catalytic and regulatory domains within a subunit and catalytic sites between subunits.
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http://dx.doi.org/10.1042/BCJ20160293DOI Listing
July 2016

Identification of the coupling step in Na(+)-translocating NADH:quinone oxidoreductase from real-time kinetics of electron transfer.

Biochim Biophys Acta 2016 Feb 4;1857(2):141-149. Epub 2015 Dec 4.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

Bacterial Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) uses a unique set of prosthetic redox groups-two covalently bound FMN residues, a [2Fe-2S] cluster, FAD, riboflavin and a Cys4[Fe] center-to catalyze electron transfer from NADH to ubiquinone in a reaction coupled with Na(+) translocation across the membrane. Here we used an ultra-fast microfluidic stopped-flow instrument to determine rate constants and the difference spectra for the six consecutive reaction steps of Vibrio harveyi Na(+)-NQR reduction by NADH. The instrument, with a dead time of 0.25 ms and optical path length of 1 cm allowed collection of visible spectra in 50-μs intervals. By comparing the spectra of reaction steps with the spectra of known redox transitions of individual enzyme cofactors, we were able to identify the chemical nature of most intermediates and the sequence of electron transfer events. A previously unknown spectral transition was detected and assigned to the Cys4[Fe] center reduction. Electron transfer from the [2Fe-2S] cluster to the Cys4[Fe] center and all subsequent steps were markedly accelerated when Na(+) concentration was increased from 20 μM to 25 mM, suggesting coupling of the former step with tight Na(+) binding to or occlusion by the enzyme. An alternating access mechanism was proposed to explain electron transfer between subunits NqrF and NqrC. According to the proposed mechanism, the Cys4[Fe] center is alternatively exposed to either side of the membrane, allowing the [2Fe-2S] cluster of NqrF and the FMN residue of NqrC to alternatively approach the Cys4[Fe] center from different sides of the membrane.
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http://dx.doi.org/10.1016/j.bbabio.2015.12.001DOI Listing
February 2016

NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase.

J Bacteriol 2015 Dec 7;198(4):655-63. Epub 2015 Dec 7.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

Unlabelled: Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na(+) translocation across the membrane. Na(+)-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na(+)-NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na(+)-NQR, resulted in an enzyme incapable of Na(+)-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na(+)-NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na(+)-NQR, which could be recovered by an nqrM-containing plasmid. The Na(+)-NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na(+)-NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na(+)-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na(+)-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE.

Importance: Na(+)-translocating NADH:quinone oxidoreductase complex (Na(+)-NQR) is a unique primary Na(+) pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio parahaemolyticus, Haemophilus influenzae, Neisseria gonorrhoeae, Pasteurella multocida, Porphyromonas gingivalis, Enterobacter aerogenes, and Yersinia pestis. Production of Na(+)-NQR in bacteria requires Na(+)-NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na(+)-NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na(+)-NQR from the Vibrio harveyi nqr operon. NqrM may be involved in Fe delivery to a unique Cys4[Fe] center during Na(+)-NQR assembly. Besides highlighting Na(+)-NQR biogenesis, these findings suggest a novel drug target to combat Na(+)-NQR-containing bacteria.
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http://dx.doi.org/10.1128/JB.00757-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751810PMC
December 2015

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

J Biol Chem 2015 Nov 23;290(46):27594-603. Epub 2015 Sep 23.

the Belozersky Institute of Physico-Chemical Biology and Department of Chemistry, Lomonosov Moscow State University, Moscow 119899, Russia

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. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.
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http://dx.doi.org/10.1074/jbc.M115.680272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646011PMC
November 2015

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

Biochem J 2015 Apr;467(2):281-91

*Department of Biochemistry, University of Turku, Turku FIN-20014, Finland.

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.
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http://dx.doi.org/10.1042/BJ20141434DOI Listing
April 2015

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

J Biol Chem 2014 Aug 1;289(33):22865-22876. Epub 2014 Jul 1.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119899, Russia. Electronic address:

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. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg(2+) except for the C. novyi PPase where Mg(2+) produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.
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http://dx.doi.org/10.1074/jbc.M114.589473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132789PMC
August 2014

Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na(+)-translocating NADH:quinone oxidoreductase.

Biochim Biophys Acta 2014 Jul 20;1837(7):1122-9. Epub 2013 Dec 20.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), and a 99.5kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na(+)-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na(+)-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na(+)-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na(+)-NQR. A new, "electron loop" mechanism is proposed for Na(+)-NQR, involving an electroneutral Na(+)/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
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http://dx.doi.org/10.1016/j.bbabio.2013.12.006DOI Listing
July 2014

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

J Biol Chem 2013 Dec 24;288(49):35489-99. Epub 2013 Oct 24.

From the Department of Biochemistry, University of Turku, FIN-20014 Turku, Finland and.

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. H(+) accumulation in vesicles was measured with fluorescent pH indicators. At pH 6.2-8.2, H(+) transport activity was high at 0.1 mM Na(+) but decreased progressively with increasing Na(+) concentrations until virtually disappearing at 5 mM Na(+). In contrast, (22)Na(+) transport activity changed little over a Na(+) concentration range of 0.05-10 mM. Conservative substitutions of gate Glu(242) and nearby Ser(243) and Asn(677) residues reduced the catalytic and transport functions of the enzyme but did not affect the Na(+) dependence of H(+) transport, whereas a Lys(681) substitution abolished H(+) (but not Na(+)) transport. All four substitutions markedly decreased PPase affinity for the activating Na(+) ion. These results are interpreted in terms of a model that assumes the presence of two Na(+)-binding sites in the channel: one associated with the gate and controlling all enzyme activities and the other located at a distance and controlling only H(+) transport activity. The inherent H(+) transport activity of Na(+)-PPase provides a rationale for its easy evolution toward specific H(+) transport.
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http://dx.doi.org/10.1074/jbc.M113.510909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853295PMC
December 2013

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

Microbiol Mol Biol Rev 2013 Jun;77(2):267-76

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia.

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). Both these transporters require Na(+) for pyrophosphate hydrolysis and are further activated by K(+). The determination of the three-dimensional structures of H(+)- and Na(+)-pyrophosphatases has been another recent breakthrough in the studies of these cation pumps. Structural and functional studies have highlighted the major determinants of the cation specificities of membrane pyrophosphatases and their potential use in constructing transgenic stress-resistant organisms.
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http://dx.doi.org/10.1128/MMBR.00003-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668671PMC
June 2013

Alternative pyrimidine biosynthesis protein ApbE is a flavin transferase catalyzing covalent attachment of FMN to a threonine residue in bacterial flavoproteins.

J Biol Chem 2013 May 4;288(20):14276-14286. Epub 2013 Apr 4.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia. Electronic address:

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na(+)-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg(2+)-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na(+)-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase.
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http://dx.doi.org/10.1074/jbc.M113.455402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3656284PMC
May 2013

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

Proc Natl Acad Sci U S A 2013 Jan 7;110(4):1255-60. Epub 2013 Jan 7.

Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.

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. However, experiments using a fluorescent pH probe and (22)Na(+) measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H(+) and Na(+) into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na(+),H(+)-PPase subfamily, which apparently evolved from Na(+)-PPases. Interestingly, genes for Na(+),H(+)-PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na(+),H(+)-PPases require Na(+) for hydrolytic and transport activities and are further activated by K(+). Based on ionophore effects, we conclude that the Na(+) and H(+) transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na(+),H(+)-PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na(+) and H(+) can be transported via the same mechanism, with switching between Na(+) and H(+) specificities requiring only subtle changes in the transporter structure.
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http://dx.doi.org/10.1073/pnas.1217816110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557053PMC
January 2013
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