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Yuxing Chen - One of the best experts on this subject based on the ideXlab platform.
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structural and enzymatic characterization of the streptococcal atp Diadenosine Polyphosphate and phosphodiester hydrolase spr1479 saph
Journal of Biological Chemistry, 2011Co-Authors: Yong-liang Jiang, Jun-wei Zhang, Wang Cheng, Chen-chen Zhang, Cecile Frolet, Anne-marie Di Guilmi, Thierry Vernet, Cong-zhao Zhou, Yuxing ChenAbstract:Abstract Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 A and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 A, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe3+ and Mn2+ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for Diadenosine Polyphosphate (ApnA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/ApnA and phosphodiester hydrolase.
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Structural and Enzymatic Characterization of the Streptococcal ATP/Diadenosine Polyphosphate and Phosphodiester Hydrolase Spr1479/SapH
Journal of Biological Chemistry, 2011Co-Authors: Yong-liang Jiang, Jun-wei Zhang, Wang Cheng, Chen-chen Zhang, Cecile Frolet, Anne-marie Di Guilmi, Thierry Vernet, Cong-zhao Zhou, Yuxing ChenAbstract:Abstract Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 A and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 A, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe3+ and Mn2+ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for Diadenosine Polyphosphate (ApnA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/ApnA and phosphodiester hydrolase.
Charles Brenner - One of the best experts on this subject based on the ideXlab platform.
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© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Fhit-nucleotide Specificity Probed with Novel Fluorescent and
2008Co-Authors: Fluorogenic Substrates, Ra Draganescu, Santosh C. Hodawadekar, Kyle R. Gee, Charles BrennerAbstract:Fhit, a member of the histidine triad superfamily of nucleotide-binding proteins, binds and cleaves Diadenosine Polyphosphates and functions as a tumor suppressor in human epithelial cancers. Function of Fhit in tumor suppression does not require Diadenosine Polyphosphate cleavage but correlates with the ability to form substrate complexes. As Diadenosine Polyphosphates are at lower cellular concentrations than mononucleotides, we sought to quantify interactions between Fhit and competitive inhibitors with the use of Diadenosine Polyphosphate analogs containing fluorophores in place of one nucleoside. Appp-S-(7-diethylamino-4methyl-3-(4-succinimidylphenyl)) coumarin (ApppAMC), Appp-S-(4-4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacine-3-yl) methylaminoacetyl (ApppBODIPY), an
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Hint, Fhit, and GalT: function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases.
Biochemistry, 2002Co-Authors: Charles BrennerAbstract:HIT (histidine triad) proteins, named for a motif related to the sequence HφHφHφφ (φ, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the α-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5‘-monophosphoramide hydrolases, the Fhit branch, which consists of Diadenosine Polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, Diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a ...
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Current Topics Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and
2002Co-Authors: Charles BrennerAbstract:ABSTRACT: HIT (histidine triad) proteins, named for a motif related to the sequence HφHφHφφ (φ, a hydrophobic amino acid), are a superfamily of nucleotide hydrolases and transferases, which act on the R-phosphate of ribonucleotides, and contain a ∼30 kDa domain that is typically either a homodimer of ∼15 kDa polypeptides with two active-sites or an internally, imperfectly repeated polypeptide that retains a single HIT active site. On the basis of sequence, substrate specificity, structure, evolution, and mechanism, HIT proteins can be classified into the Hint branch, which consists of adenosine 5′-monophosphoramide hydrolases, the Fhit branch, which consists of Diadenosine Polyphosphate hydrolases, and the GalT branch, which consists of specific nucleoside monophosphate transferases, including galactose-1-phosphate uridylyltransferase, Diadenosine tetraphosphate phosphorylase, and adenylyl sulfate:phosphate adenylytransferase. At least one human representative of each branch is lost in human diseases. Aprataxin, a Hint branch hydrolase, is mutated in ataxia-oculomotor apraxia syndrome. Fhit is lost early in the development of many epithelially derived tumors. GalT is deficient in galactosemia. Additionally, ASW is an avian Hint family member that has evolved to have unusual gene expression properties and the complete loss of its nucleotide binding site. The potential roles of ASW and Hint in avian sexual development are discussed elsewhere. Here we review what is known about biological activities of HIT proteins, the structura
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Di-, tri- and tetra-5'-O-phosphorothioadenosyl substituted polyols as inhibitors of Fhit: Importance of the α-β bridging oxygen and β phosphorus replacement
BMC Chemical Biology, 2001Co-Authors: James M Varnum, Wojciech J. Stec, Janina Baraniak, Renata Kaczmarek, Charles BrennerAbstract:Background The human FHIT gene is inactivated early in the development of many human cancers and loss of Fhit in mouse predisposes to cancer while reintroduction of FHIT suppresses tumor formation via induction of apoptosis. Fhit protein, a Diadenosine Polyphosphate hydrolase, does not require hydrolase activity to function in tumor suppression and may signal for apoptosis as an enzyme-substrate complex. Thus, high affinity nonhydrolyzable substrate analogs may either promote or antagonize Fhit function, depending on their features, in Fhit + cells. Previously synthesized analogs with phosphorothioadenosyl substitutions and "supercharged" branches do not bind better than natural substrates and thus have limited potential as cellular probes. Results Here we link adenosine 5'- O -phosphates and phosphorothioates to short-chain polyols to generate a series of substrate analogs. We obtain structure-activity data in the form of in vitro Fhit inhibition for four types of analog substitutions and describe two compounds, inhibitory constants for which are 65 and 75-fold lower than natural substrates. Conclusions The best Fhit inhibitors obtained to date separate two or more 5'- O -phosphoromonothioadenosyl moieties with as many bond lengths as in AppppA, maintain oxygen at the location of the α-β bridging oxygen, and replace carbon for the β phosphorus.
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Crystal structures of HINT demonstrate that histidine triad proteins are GalT-related nucleotide-binding proteins
Nature Structural Biology, 1997Co-Authors: Charles Brenner, Preston Garrison, Jeffrey Gilmour, Daniel Peisach, Dagmar Ringe, Gregory A. Petsko, John M. LowensteinAbstract:Histidine triad nucleotide-binding protein (HINT), a dimeric purine nucleotide-binding protein from rabbit heart, is a member of the HIT (histidine triad) superfamily which includes HINT homologues and FHIT (HIT protein encoded at the chromosome 3 fragile site) homologues. Crystal structures of HINT-nucleotide complexes demonstrate that the most conserved residues in the superfamily mediate nucleotide binding and that the HIT motif forms part of the phosphate binding loop. Galactose-1-phosphate uridylyltransferase, whose deficiency causes galactosemia, contains tandem HINT domains with the same fold and mode of nucleotide binding as HINT despite having no overall sequence similarity. Features of FHIT, a Diadenosine Polyphosphate hydrolase and candidate tumour suppressor, are predicted from HINT-nucleotide structures.
Andrew D Miller - One of the best experts on this subject based on the ideXlab platform.
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biotin c10 appch2ppa is an effective new chemical proteomics probe for Diadenosine Polyphosphate binding proteins
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Ameruddin M Azhar, Ahmed Kamal, Andrew D Miller, Michael Wright, Judith NagyAbstract:Here we report on the synthesis of a synthetic, stable biotin-c10-AppCH2ppA conjugate involving an unusual Cannizzaro reaction step. This conjugate is used to bind prospective Ap4A binding proteins from Escherichia coli bacterial cell lyzates. Following binding, identities of these proteins are then determined smoothly by a process of magnetic bio-panning and electrospray mass spectrometry. Protein hits appear to be a definitive set of stress protein related targets. While this hit list may not be exclusive, and may vary with the nature of sampling conditions and organism status, nevertheless hits do appear to correspond with bona fide Ap4A-binding proteins. Therefore these hits represent a sound basis on which to construct new hypotheses concerning the cellular importance of Ap4A to bacterial cells and the potential biological significance of Ap4A-protein binding interactions.
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syntheses of stable synthetic Diadenosine Polyphosphate analogues using recombinant histidine tagged lysyl trna synthetase lysu
Bioorganic & Medicinal Chemistry Letters, 2014Co-Authors: Mike Wright, Ameruddin M Azhar, Ahmed Kamal, Andrew D MillerAbstract:Abstract Recombinant Escherichia coli lysyl-tRNA synthase (LysU) has been previously utilised in the production of stabile, synthetic Diadenosine Polyphosphate (Ap n A) analogues. Here we report on the extended use of a new recombinant histidine residue-tagged LysU as a tool for highly controlled phosphate phosphate bond formation between nucleotides, avoiding the need for complex protecting group chemistries. Resulting high yielding tandem LysU-based biosynthetic–synthetic/synthetic–biosynthetic strategies emerge for the preparation of varieties of Ap n A analogues directly from inexpensive natural nucleotides and nucleosides. Analogues so formed make a useful small library with which to probe Ap n A activities in vitro and in vivo leading to the discovery of new, potentially potent biopharmaceuticals active against chronic pain and other chronic, high-burden disease states.
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Diadenosine Polyphosphate analog controls postsynaptic excitation in ca3 ca1 synapses via a nitric oxide dependent mechanism
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Sergei Melnik, Mike Wright, Andrew D Miller, J A Tanner, Timur Tsintsadze, Vera Tsintsadze, Natalia LozovayaAbstract:Previously, we have described the modulatory effect of Diadenosine Polyphosphates Ap4A and Ap5A on synaptic transmission in the rat hippocampal slices mediated by presynaptic receptors ([Klishin et al., 1994][1]). In contrast, we now describe how nonhydrolyzable Ap4A analog Diadenosine-5′,5′′′- P 1, P 4-[β,β′-methylene]tetraphosphate (AppCH2ppA) at low micromolar concentrations exerts strong nondesensitizing inhibition of orthodromically evoked field potentials (OFPs) without affecting the amplitude of excitatory postsynaptic currents and antidromically evoked field potentials, as recorded in hippocampal CA1 zone. The effects of AppCH2ppA on OFPs are eliminated by a P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) but not mimicked by purinoceptor agonists α,β-methylene-ATP and adenosine 5′- O -(3-thio)-triphosphate, indicating that a P2-like receptor is involved but not one belonging to the conventional P2X/P2Y receptor classes. Diadenosine Polyphosphate receptor (P4) antagonist Ip4I (diinosine tetraphosphate) was unable to modulate AppCH2ppA effects. Thus, the PPADS-sensitive P2-like receptor for AppCH2ppA seems to control selectively dendritic excitation of the CA1 neurons. The specific nitric oxide (NO)-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide is shown to significantly attenuate AppCH2ppA-mediated inhibitory effects, indicating that NO is involved in the cascade of events initiated by AppCH2ppA. Further downstream mediation by adenosine A1 receptors is also demonstrated. Hence, AppCH2ppA-mediated effects involve PPADS-sensitive P2-like receptor activation leading to the production of NO that stimulates intracellular synthesis of adenosine, causing in turn postsynaptic A1 receptor activation and subsequent postsynaptic CA1 dendritic inhibition. Such spatially selective postsynaptic dendritic inhibition may influence dendritic electrogenesis in pyramidal neurons and consequently mediate control of neuronal network activity. [1]: #ref-42
Pedro Rotllan - One of the best experts on this subject based on the ideXlab platform.
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biochemical analysis of ecto nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of npp1 isoenzyme in extracellular hydrolysis of Diadenosine Polyphosphates in central nervous system
Neurochemistry International, 2007Co-Authors: Aaron C. Asensio, Carmen R Rodriguezferrer, Agustin Castaneyraperdomo, Sol Oaknin, Pedro RotllanAbstract:Abstract Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides Diadenosine Polyphosphates. Intact synaptosomes and plasma and synaptic membranes isolated by sucrose-gradient ultracentrifugation from several brain regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum and cerebellum) degraded the fluorogenic substrates diethenoadenosine Polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved substrates always releasing the mononucleotide moieties ethenoadenosine 5′-monophosphate and the corresponding ethenoadenosine (n − 1) 5′-phosphate. Ectoenzymatic hydrolysis reached maximal activity at pH 9.0 (pH range 6.5–9.0) and was activated by Ca2+ and Mg2+ ions, with maximal effects around 2.0 mM cation. EDTA drastically reduced activity and Zn2+ was required for enzyme reactivation. Hydrolysis of substrates followed hyperbolic kinetics with Km values in the 3–10 μM range. Diadenosine Polyphosphates and heparin behaved as competitive inhibitors in the enzymatic hydrolysis of diethenoadenosine Polyphosphates and AMP, ATP, α,β-methyleneADP, ADPβS ATPγS, β,γ-methyleneATP, suramin and diethyl pyrocarbonate were also inhibitors. Ectoenzymatic activity shared the typical characteristics of members of the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family and inhibition data suggest that NPP1 ectoenzyme is involved in the cleavage of extracellular Diadenosine Polyphosphates in brain. Synaptic membranes from cerebellum, hypothalamus and hippocampus presented the highest activities and no activity differences were observed between young and aged animals. However, plasma membranes showed a more homogeneous distribution of ectoenzymatic activity but a general increase was detected in aged animals. Enhancement of ectoenzymatic Diadenosine Polyphosphate cleaving activity found in plasma membranes from old animals could play a deleterious role in aged brain by limiting neuroprotective effects reported for extracellular Diadenosine tetraphosphate.
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Biochemical analysis of ecto-nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of NPP1 isoenzyme in extracellular hydrolysis of Diadenosine Polyphosphates in central nervous system
Neurochemistry International, 2007Co-Authors: Aaron C. Asensio, Carmen R. Rodríguez-ferrer, Sol Oaknin, Agustín Castañeyra-perdomo, Pedro RotllanAbstract:Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides Diadenosine Polyphosphates. Intact synaptosomes and plasma and synaptic membranes isolated by sucrose-gradient ultracentrifugation from several brain regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum and cerebellum) degraded the fluorogenic substrates diethenoadenosine Polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved substrates always releasing the mononucleotide moieties ethenoadenosine 5'-monophosphate and the corresponding ethenoadenosine (n-1) 5'-phosphate. Ectoenzymatic hydrolysis reached maximal activity at pH 9.0 (pH range 6.5-9.0) and was activated by Ca(2+) and Mg(2+) ions, with maximal effects around 2.0 mM cation. EDTA drastically reduced activity and Zn(2+) was required for enzyme reactivation. Hydrolysis of substrates followed hyperbolic kinetics with K(m) values in the 3-10 microM range. Diadenosine Polyphosphates and heparin behaved as competitive inhibitors in the enzymatic hydrolysis of diethenoadenosine Polyphosphates and AMP, ATP, alpha,beta-methyleneADP, ADPbetaS ATPgammaS, beta,gamma-methyleneATP, suramin and diethyl pyrocarbonate were also inhibitors. Ectoenzymatic activity shared the typical characteristics of members of the ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family and inhibition data suggest that NPP1 ectoenzyme is involved in the cleavage of extracellular Diadenosine Polyphosphates in brain. Synaptic membranes from cerebellum, hypothalamus and hippocampus presented the highest activities and no activity differences were observed between young and aged animals. However, plasma membranes showed a more homogeneous distribution of ectoenzymatic activity but a general increase was detected in aged animals. Enhancement of ectoenzymatic Diadenosine Polyphosphate cleaving activity found in plasma membranes from old animals could play a deleterious role in aged brain by limiting neuroprotective effects reported for extracellular Diadenosine tetraphosphate.
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Potent inhibition of specific Diadenosine Polyphosphate hydrolases by suramin
FEBS Letters, 1998Co-Authors: Pedro Rotllan, Carmen R. Rodríguez-ferrer, Aaron C. Asensio, Sol OakninAbstract:Abstract The cytosolic enzymes asymmetrical Diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap4Aase) and Diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap3Aase) are inhibited competitively by suramin. Ap4Aase and Ap3Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates Diadenosine tetraphosphate (Ap4A) and Diadenosine triphosphate (Ap3A). Ki values for suramin as inhibitor of Ap4Aase and Ap3Aase were 5×10−6 M and 3×10−7 M, respectively. Results indicate that suramin or suramin-like derivatives may be useful tools to investigate Diadenosine Polyphosphate cleaving enzymes and that the intracellular Diadenosine Polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.
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Potent inhibition of specific Diadenosine Polyphosphate hydrolases by suramin
FEBS Letters, 1998Co-Authors: Pedro Rotllan, Carmen R. Rodríguez-ferrer, Aaron C. Asensio, Sol OakninAbstract:Abstract The cytosolic enzymes asymmetrical Diadenosine tetraphosphate hydrolase (EC 3.6.1.17, Ap4Aase) and Diadenosine triphosphate hydrolase (EC 3.6.1.29, Ap3Aase) are inhibited competitively by suramin. Ap4Aase and Ap3Aase were assayed in cytosolic rat brain extracts using fluorogenic analogues of the respective substrates Diadenosine tetraphosphate (Ap4A) and Diadenosine triphosphate (Ap3A). Ki values for suramin as inhibitor of Ap4Aase and Ap3Aase were 5×10−6 M and 3×10−7 M, respectively. Results indicate that suramin or suramin-like derivatives may be useful tools to investigate Diadenosine Polyphosphate cleaving enzymes and that the intracellular Diadenosine Polyphosphate metabolism may be a pharmacological target of suramin with biological and clinical implications.
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suramin a powerful inhibitor of neural ecto Diadenosine Polyphosphate hydrolase
British Journal of Pharmacology, 1996Co-Authors: Jesús Mateo, Pedro Rotllan, Maria Teresa MirasportugalAbstract:The neural ecto-Diadenosine Polyphosphate hydrolase (ecto-ApnAase) from plasma membranes of Torpedo synaptic terminals is inhibited by suramin. This study was carried out by discontinuous h.p.l.c. and continuous fluorometric methods. The concentration-dependence studies showed a non-competitive mechanism for suramin in the Dixon plot, with a Ki value of 1.79 +/- 0.03 microM with respect to epsilon-(Ap3A) as the substrate and 1.69 +/- 0.05 microM and 1.86 +/- 0.06 microM for epsilon-(Ap4A) and epsilon-(Ap5A) respectively. These results indicate that suramin could be a base compound inhibiting ecto-ApnAase and providing an alternative way of studying the pharmacology of Diadenosine Polyphosphate receptors.
Yong-liang Jiang - One of the best experts on this subject based on the ideXlab platform.
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structural and enzymatic characterization of the streptococcal atp Diadenosine Polyphosphate and phosphodiester hydrolase spr1479 saph
Journal of Biological Chemistry, 2011Co-Authors: Yong-liang Jiang, Jun-wei Zhang, Wang Cheng, Chen-chen Zhang, Cecile Frolet, Anne-marie Di Guilmi, Thierry Vernet, Cong-zhao Zhou, Yuxing ChenAbstract:Abstract Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 A and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 A, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe3+ and Mn2+ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for Diadenosine Polyphosphate (ApnA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/ApnA and phosphodiester hydrolase.
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Structural and Enzymatic Characterization of the Streptococcal ATP/Diadenosine Polyphosphate and Phosphodiester Hydrolase Spr1479/SapH
Journal of Biological Chemistry, 2011Co-Authors: Yong-liang Jiang, Jun-wei Zhang, Wang Cheng, Chen-chen Zhang, Cecile Frolet, Anne-marie Di Guilmi, Thierry Vernet, Cong-zhao Zhou, Yuxing ChenAbstract:Abstract Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 A and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 A, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe3+ and Mn2+ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for Diadenosine Polyphosphate (ApnA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/ApnA and phosphodiester hydrolase.