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Alfred Pingoud - One of the best experts on this subject based on the ideXlab platform.
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Cleavage experiments with deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) suggest that the homing endonuclease I-PpoI follows the same mechanism of Phosphodiester Bond hydrolysis as the non-specific Serratia nuclease1
FEBS Letters, 1999Co-Authors: Peter Friedhoff, Ingo Franke, Kurt L. Krause, Alfred PingoudAbstract:We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg2+ bound to an asparagine residue as a Lewis acid in Phosphodiester Bond cleavage.
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cleavage experiments with deoxythymidine 3 5 bis p nitrophenyl phosphate suggest that the homing endonuclease i ppoi follows the same mechanism of Phosphodiester Bond hydrolysis as the non specific serratia nuclease1
FEBS Letters, 1999Co-Authors: Peter Friedhoff, Ingo Franke, Kurt L. Krause, Alfred PingoudAbstract:We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg2+ bound to an asparagine residue as a Lewis acid in Phosphodiester Bond cleavage.
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Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease
FEBS Journal, 1996Co-Authors: Peter Friedhoff, Gregor Meiss, Bettina Kolmes, Uwe Pieper, Oleg Gimadutdinow, Claus Urbanke, Alfred PingoudAbstract:The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile Phosphodiester Bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent Phosphodiester Bond. Furthermore, the modified Bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified Phosphodiester Bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the Phosphodiester Bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p-nitrophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-(p-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.
Peter Friedhoff - One of the best experts on this subject based on the ideXlab platform.
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Cleavage experiments with deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) suggest that the homing endonuclease I-PpoI follows the same mechanism of Phosphodiester Bond hydrolysis as the non-specific Serratia nuclease1
FEBS Letters, 1999Co-Authors: Peter Friedhoff, Ingo Franke, Kurt L. Krause, Alfred PingoudAbstract:We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg2+ bound to an asparagine residue as a Lewis acid in Phosphodiester Bond cleavage.
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cleavage experiments with deoxythymidine 3 5 bis p nitrophenyl phosphate suggest that the homing endonuclease i ppoi follows the same mechanism of Phosphodiester Bond hydrolysis as the non specific serratia nuclease1
FEBS Letters, 1999Co-Authors: Peter Friedhoff, Ingo Franke, Kurt L. Krause, Alfred PingoudAbstract:We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3′,5′-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg2+ bound to an asparagine residue as a Lewis acid in Phosphodiester Bond cleavage.
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Kinetic Analysis of the Cleavage of Natural and Synthetic Substrates by the Serratia Nuclease
FEBS Journal, 1996Co-Authors: Peter Friedhoff, Gregor Meiss, Bettina Kolmes, Uwe Pieper, Oleg Gimadutdinow, Claus Urbanke, Alfred PingoudAbstract:The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile Phosphodiester Bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent Phosphodiester Bond. Furthermore, the modified Bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified Phosphodiester Bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the Phosphodiester Bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p-nitrophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-(p-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.
Pierre-alain Monnard - One of the best experts on this subject based on the ideXlab platform.
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formation of rna Phosphodiester Bond by histidine containing dipeptides
ChemBioChem, 2013Co-Authors: Rafal Wieczorek, Mark Dörr, Agata Chotera, Pier Luigi Luisi, Pierre-alain MonnardAbstract:A new scenario for prebiotic formation of nucleic acid oligomers is presented. Peptide catalysis is applied to achieve condensation of activated RNA monomers into short RNA chains. As catalysts, L-dipeptides containing a histidine residue, primarily Ser-His, were used. Reactions were carried out in self-organised environment, a water-ice eutectic phase, with low concentrations of reactants. Incubation periods up to 30 days resulted in the formation of short oligomers of RNA. During the oligomerisation, an active intermediate (dipeptide-mononucleotide) is produced, which is the reactive species. Details of the mechanism and kinetics, which were elucidated with a set of control experiments, further establish that the imidazole side chain of a histidine at the carboxyl end of the dipeptide plays a crucial role in the catalysis. These results suggest that this oligomerisation catalysis occurs by a transamination mechanism. Because peptides are much more likely products of spontaneous condensation than nucleotide chains, their potential as catalysts for the formation of RNA is interesting from the origin-of-life perspective. Finally, the formation of the dipeptide-mononucleotide intermediate and its significance for catalysis might also be viewed as the tell-tale signs of a new example of organocatalysis.
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Formation of RNA Phosphodiester Bond by Histidine‐Containing Dipeptides
ChemBioChem, 2012Co-Authors: Rafal Wieczorek, Mark Dörr, Agata Chotera, Pier Luigi Luisi, Pierre-alain MonnardAbstract:A new scenario for prebiotic formation of nucleic acid oligomers is presented. Peptide catalysis is applied to achieve condensation of activated RNA monomers into short RNA chains. As catalysts, L-dipeptides containing a histidine residue, primarily Ser-His, were used. Reactions were carried out in self-organised environment, a water-ice eutectic phase, with low concentrations of reactants. Incubation periods up to 30 days resulted in the formation of short oligomers of RNA. During the oligomerisation, an active intermediate (dipeptide-mononucleotide) is produced, which is the reactive species. Details of the mechanism and kinetics, which were elucidated with a set of control experiments, further establish that the imidazole side chain of a histidine at the carboxyl end of the dipeptide plays a crucial role in the catalysis. These results suggest that this oligomerisation catalysis occurs by a transamination mechanism. Because peptides are much more likely products of spontaneous condensation than nucleotide chains, their potential as catalysts for the formation of RNA is interesting from the origin-of-life perspective. Finally, the formation of the dipeptide-mononucleotide intermediate and its significance for catalysis might also be viewed as the tell-tale signs of a new example of organocatalysis.
Wei Yang - One of the best experts on this subject based on the ideXlab platform.
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watching dna polymerase η make a Phosphodiester Bond
Nature, 2012Co-Authors: Teruya Nakamura, Ye Zhao, Yuriko Yamagata, Wei YangAbstract:DNA synthesis has been extensively studied, but the chemical reaction itself has not been visualized. Here we follow the course of Phosphodiester Bond formation using time-resolved X-ray crystallography. Native human DNA polymerase η, DNA and dATP were co-crystallized at pH 6.0 without Mg2+. The polymerization reaction was initiated by exposing crystals to 1 mM Mg2+ at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg2+ ions are aligned within 40 s, but the Bond formation is not evident until 80 s. From 80 to 300 s structures show a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicate that deprotonation and an accompanying C2′-endo to C3′-endo conversion of the nucleophile 3′-OH are rate limiting. A third Mg2+ ion, which arrives with the new Bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism. Atomic-resolution time courses of Phosphodiester Bond formation catalysed by DNA polymerase η reveal transient intermediate states and an unexpected third metal ion in the reaction mechanism. Chemists would like to be able to determine the structures of true transition states in chemical reactions, but the high energy and unstable nature of transition states had made this goal unattainable. Using a repair reaction catalysed by DNA polymerase η (Pol η) as their model, Wei Yang and colleagues have extended the use of flash–freeze technology to observe DNA synthesis in real time and at atomic resolution using X-ray crystallography to analyse the trapped covalent intermediates. Pol η is particularly well suited to this approach because it has a slow rate of reaction and a relatively rigid catalytic centre. The observed reaction intermediates reveal several unanticipated transient states, and implicate an unexpected third magnesium ion in the reaction mechanism.
Jean-pierre Perreault - One of the best experts on this subject based on the ideXlab platform.
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Peach latent mosaic viroid is locked by a 2′,5′-Phosphodiester Bond produced by in vitro self-ligation.
Journal of Molecular Biology, 1997Co-Authors: F. Côté, Jean-pierre PerreaultAbstract:Abstract Although some viroid-like satellite RNAs possess self-cleavage and self-ligation activities, we show that the peach latent mosaic viroid (PLMVd) is unique among all known viroids since it also has such activities. These catalytic activities should have important roles in the rolling circle replication of PLMVd. According to this proposed mechanism, self-cleavage of the multimeric strands occurs via hammerhead structures producing monomers possessing 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In the most stable predicted secondary structure for PLMVd these two extremities are juxtaposed, in order for self-ligation to occur. To establish the nature of the Phosphodiester Bond produced by self-ligation, we followed the classical procedure of complete enzymatic RNA hydrolysis coupled with thin layer chromatography fractionation. Using this procedure, we report that the self-ligation of PLMVd transcripts produces almost exclusively the 2′,5′ isomer (>96%). Primer extension assays also revealed that reverse transcriptase can read througth this 2′,5′ linkage, suggesting that it does not prevent further replication of the viroid. Moreover, we have observed that this 2′,5′ linkage is resistant to the debranching activity contained in nuclear extracts, as well as being capable of preventing further viroid self-cleavage. Thus, if viroids do indeed self-ligate in vivo , the resulting 2′,5′-Phosphodiester Bond could contribute to the stability of these RNA species. Finally, an analysis of both the sequence and the structural requirements for hammerhead self-cleavage and self-ligation suggests that these two RNA processes may be interrelated. We hypothesize that the intramolecular self-ligation which produces circular conformers may contribute to the circularization step of the rolling circle replication, while the intermolecular non-enzymatic ligation is a potential mechanism for the sequence reassortment of viroids and viroid-like species.
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peach latent mosaic viroid is locked by a 2 5 Phosphodiester Bond produced by in vitro self ligation
Journal of Molecular Biology, 1997Co-Authors: F. Côté, Jean-pierre PerreaultAbstract:Abstract Although some viroid-like satellite RNAs possess self-cleavage and self-ligation activities, we show that the peach latent mosaic viroid (PLMVd) is unique among all known viroids since it also has such activities. These catalytic activities should have important roles in the rolling circle replication of PLMVd. According to this proposed mechanism, self-cleavage of the multimeric strands occurs via hammerhead structures producing monomers possessing 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In the most stable predicted secondary structure for PLMVd these two extremities are juxtaposed, in order for self-ligation to occur. To establish the nature of the Phosphodiester Bond produced by self-ligation, we followed the classical procedure of complete enzymatic RNA hydrolysis coupled with thin layer chromatography fractionation. Using this procedure, we report that the self-ligation of PLMVd transcripts produces almost exclusively the 2′,5′ isomer (>96%). Primer extension assays also revealed that reverse transcriptase can read througth this 2′,5′ linkage, suggesting that it does not prevent further replication of the viroid. Moreover, we have observed that this 2′,5′ linkage is resistant to the debranching activity contained in nuclear extracts, as well as being capable of preventing further viroid self-cleavage. Thus, if viroids do indeed self-ligate in vivo , the resulting 2′,5′-Phosphodiester Bond could contribute to the stability of these RNA species. Finally, an analysis of both the sequence and the structural requirements for hammerhead self-cleavage and self-ligation suggests that these two RNA processes may be interrelated. We hypothesize that the intramolecular self-ligation which produces circular conformers may contribute to the circularization step of the rolling circle replication, while the intermolecular non-enzymatic ligation is a potential mechanism for the sequence reassortment of viroids and viroid-like species.