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Reijo Lahti - One of the best experts on this subject based on the ideXlab platform.
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The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding.
Archives of biochemistry and biophysics, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Abstract 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 Co2+, 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. Co2+ 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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the tetrameric structure of nucleotide regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding
bioRxiv, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, V N Orlov, Alexander A BaykovAbstract:A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine beta-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 Co2+, 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. Co2+ 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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Cooperativity in catalysis by canonical family II pyrophosphatases
Biochemical and biophysical research communications, 2019Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Vera A. Aksenova, Alexander A BaykovAbstract:Abstract 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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An arginine residue involved in allosteric regulation of cystathionine β-synthase (CBS) Domain-containing pyrophosphatase.
Archives of biochemistry and biophysics, 2018Co-Authors: Viktor A Anashkin, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Inorganic pyrophosphatase containing a pair of regulatory CBS Domains (CBS-PPase1) 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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Inorganic pyrophosphatases of Family II-two decades after their discovery.
FEBS letters, 2017Co-Authors: Alexander A Baykov, Viktor A Anashkin, Anu Salminen, Reijo LahtiAbstract:Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) 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 (kcat ≈ 104 s-1 ) 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, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).
Alexander A Baykov - One of the best experts on this subject based on the ideXlab platform.
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The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding.
Archives of biochemistry and biophysics, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Abstract 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 Co2+, 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. Co2+ 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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the tetrameric structure of nucleotide regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding
bioRxiv, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, V N Orlov, Alexander A BaykovAbstract:A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine beta-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 Co2+, 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. Co2+ 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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Cooperativity in catalysis by canonical family II pyrophosphatases
Biochemical and biophysical research communications, 2019Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Vera A. Aksenova, Alexander A BaykovAbstract:Abstract 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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An arginine residue involved in allosteric regulation of cystathionine β-synthase (CBS) Domain-containing pyrophosphatase.
Archives of biochemistry and biophysics, 2018Co-Authors: Viktor A Anashkin, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Inorganic pyrophosphatase containing a pair of regulatory CBS Domains (CBS-PPase1) 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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Inorganic pyrophosphatases of Family II-two decades after their discovery.
FEBS letters, 2017Co-Authors: Alexander A Baykov, Viktor A Anashkin, Anu Salminen, Reijo LahtiAbstract:Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) 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 (kcat ≈ 104 s-1 ) 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, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).
Luis Alfonso Martínez-cruz - One of the best experts on this subject based on the ideXlab platform.
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Current Structural Knowledge on the CNNM Family of Magnesium Transport Mediators.
International journal of molecular sciences, 2019Co-Authors: Paula Giménez-mascarell, Iker Oyenarte, Dominik N. Müller, María L. Martínez-chantar, Irene González-recio, Carmen Fernández-rodríguez, Luis Alfonso Martínez-cruzAbstract:The cyclin and cystathionine β-synthase (CBS) Domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg2+/Co2+ efflux protein CorC and the Mg2+ channel MgtE.
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Current Structural Knowledge on the CNNM Family of Magnesium Transport Mediators
MDPI AG, 2019Co-Authors: Paula Giménez-mascarell, Iker Oyenarte, María Luz Martínez-chantar, Irene González-recio, Carmen Fernández-rodríguez, Dominik Müller, Luis Alfonso Martínez-cruzAbstract:The cyclin and cystathionine β-synthase (CBS) Domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg2+/Co2+ efflux protein CorC and the Mg2+ channel MgtE
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CBS Domains: Ligand binding sites and conformational variability.
Archives of biochemistry and biophysics, 2013Co-Authors: June Ereño-orbea, Iker Oyenarte, Luis Alfonso Martínez-cruzAbstract:Cystathionine β-synthase (CBS) Domains or CBS motifs are conserved structural Domains that are present in thousands of non functionally-related proteins from all kingdoms of life. Their importance is underlined by the range of hereditary diseases associated with mutations in their amino acid sequence. CBS motifs associate in pairs referred to as Bateman modules. In contrast with initial assumptions, it is now well documented that CBS motifs and/or Bateman modules may suffer conformational changes upon binding of adenosine derivatives, metal ions or nucleic acids. The degree and direction of these structural changes depend on the type of ligand, the intrinsic features of the binding sites and the association manner of the Bateman modules. This review aims to provide a summary of the current knowledge on the structural basis of ligand recognition and on the structural effects caused by these ligands in CBS Domain containing proteins.
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Purification, crystallization and preliminary crystallographic analysis of the CBS-Domain pair of cyclin M2 (CNNM2)
Acta Crystallographica Section F Structural Biology and Crystallization Communications, 2012Co-Authors: Inmaculada Gómez-garcía, Iker Oyenarte, Marchel Stuiver, June Ereño, María Angeles Corral-rodríguez, Dominik N. Müller, Luis Alfonso Martínez-cruzAbstract:This work describes the purification and preliminary crystallographic analysis of the CBS-Domain pair of the murine CNNM2 magnesium transporter (formerly known as ancient Domain protein 2; ACDP2), which consists of a pair of cystathionine β-synthase (CBS) motifs and has 100% sequence identity to its human homologue. CNNM proteins represent the least-studied members of the eight different types of magnesium transporters identified to date in mammals. In humans, the CNNM family is encoded by four genes: CNNM1–4. CNNM1 acts as a cytosolic copper chaperone, whereas CNNM2 and CNNM4 have been associated with magnesium handling. Interestingly, mutations in the CNNM2 gene cause familial dominant hypomagnesaemia (MIM:607803), a rare human disorder characterized by renal and intestinal magnesium (Mg2+) wasting, which may lead to symptoms of Mg2+ depletion such as tetany, seizures and cardiac arrhythmias. This manuscript describes the preliminary crystallographic analysis of two different crystal habits of a truncated form of the protein containing its regulatory CBS-Domain pair, which has been reported to host the pathological mutation T568I in humans. The crystals belonged to space groups P21212 and I222 (or I212121) and diffracted X-rays to 2.0 and 3.6 A resolution, respectively, using synchrotron radiation.
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Purification, crystallization and preliminary crystallographic analysis of the CBS-Domain protein MJ1004 from Methanocaldococcus jannaschii.
Acta crystallographica. Section F Structural biology and crystallization communications, 2011Co-Authors: Iker Oyenarte, Inmaculada Gómez-garcía, María Lucas, Luis Alfonso Martínez-cruzAbstract:The purification and preliminary crystallographic analysis of the archaeal CBS-Domain protein MJ1004 from Methanocaldococcus jannaschii are described. The native protein was overexpressed, purified and crystallized in the monoclinic space group P2(1), with unit-cell parameters a=54.4, b=53.8, c=82.6 Å, β=106.1°. The crystals diffracted X-rays to 2.7 Å resolution using synchrotron radiation. Matthews-volume calculations suggested the presence of two molecules in the asymmetric unit that are likely to correspond to a dimeric species, which is also observed in solution.
Viktor A Anashkin - One of the best experts on this subject based on the ideXlab platform.
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The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding.
Archives of biochemistry and biophysics, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Abstract 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 Co2+, 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. Co2+ 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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the tetrameric structure of nucleotide regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding
bioRxiv, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, V N Orlov, Alexander A BaykovAbstract:A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine beta-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 Co2+, 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. Co2+ 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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Cooperativity in catalysis by canonical family II pyrophosphatases
Biochemical and biophysical research communications, 2019Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Vera A. Aksenova, Alexander A BaykovAbstract:Abstract 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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An arginine residue involved in allosteric regulation of cystathionine β-synthase (CBS) Domain-containing pyrophosphatase.
Archives of biochemistry and biophysics, 2018Co-Authors: Viktor A Anashkin, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Inorganic pyrophosphatase containing a pair of regulatory CBS Domains (CBS-PPase1) 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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Inorganic pyrophosphatases of Family II-two decades after their discovery.
FEBS letters, 2017Co-Authors: Alexander A Baykov, Viktor A Anashkin, Anu Salminen, Reijo LahtiAbstract:Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) 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 (kcat ≈ 104 s-1 ) 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, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).
Anu Salminen - One of the best experts on this subject based on the ideXlab platform.
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The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding.
Archives of biochemistry and biophysics, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Victor N Orlov, Alexander A BaykovAbstract:Abstract 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 Co2+, 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. Co2+ 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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the tetrameric structure of nucleotide regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding
bioRxiv, 2020Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, V N Orlov, Alexander A BaykovAbstract:A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine beta-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 Co2+, 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. Co2+ 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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Cooperativity in catalysis by canonical family II pyrophosphatases
Biochemical and biophysical research communications, 2019Co-Authors: Viktor A Anashkin, Anu Salminen, Reijo Lahti, Vera A. Aksenova, Alexander A BaykovAbstract:Abstract 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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Inorganic pyrophosphatases of Family II-two decades after their discovery.
FEBS letters, 2017Co-Authors: Alexander A Baykov, Viktor A Anashkin, Anu Salminen, Reijo LahtiAbstract:Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) 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 (kcat ≈ 104 s-1 ) 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, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).
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an asparagine residue mediates intramolecular communication in nucleotide regulated pyrophosphatase
Biochemical Journal, 2016Co-Authors: Viktor A Anashkin, Anu Salminen, Natalia N Vorobjeva, Reijo Lahti, Alexander A BaykovAbstract:Many prokaryotic soluble pyrophosphatases (PPases) contain a pair of regulatory adenine nucleotide-binding CBS Domains that act as "internal inhibitors" whose effect is modulated by nucleotide binding. Though such regulatory Domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We previously reported that CBS Domains bind nucleotides cooperatively and induce positive kinetic cooperativity (non-Michaelian behavior) in CBS Domain-containing PPases (CBS-PPases). Here, we demonstrate that a homodimeric Ethanoligenens harbinense CBS-PPase ( eh PPase) containing an inherent mutation in an otherwise conserved Asn residue in a loop near the active site exhibits non-cooperative hydrolysis kinetics. A similar Asn312Ser substitution in "cooperative" Desulfitobacterium hafniense CBS-PPase ( dh PPase) abolished kinetic cooperativity while causing only minor effects on nucleotide-binding affinity and cooperativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic cooperativity in wild-type dh PPase but restoring it in the variant dh PPase. A reverse Ser-to-Asn replacement restored kinetic cooperativity in eh PPase. Molecular dynamics simulations revealed that the Asn substitution resulted in a change in the H-bonding pattern around the Asn residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this Asn residue as lying at the "crossroads" of information paths connecting catalytic and regulatory Domains within a subunit and catalytic sites between subunits.