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Nicolas Glansdorff - One of the best experts on this subject based on the ideXlab platform.
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New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and Carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate
Hindawi Limited, 2005Co-Authors: Jan Massant, Nicolas GlansdorffAbstract:A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and Ornithine Carbamoyltransferase (OTC) from Pyrococcus furiosus was provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate Carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein–protein interactions in the physiology of hyperthermophiles
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metabolic enzymes from psychrophilic bacteria challenge of adaptation to low temperatures in Ornithine Carbamoyltransferase from moritella abyssi
Journal of Bacteriology, 2003Co-Authors: Georges Feller, Charles Gerday, Nicolas GlansdorffAbstract:The enzyme Ornithine Carbamoyltransferase (OTCase) of Moritella abyssi (OTCaseMab), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCaseMab displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCaseMab displays higher Km values for Ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue δ-N-phosphonoacetyl-l-Ornithine (PALO). OTCaseMab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high Km values and the lower sensitivity to PALO. The Km for Ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (kcat/Km) of OTCases adapted to different temperatures showed that OTCaseMab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the Km value for Ornithine observed when the temperature is brought from 20 to 5°C. OTCaseMab adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.
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metabolic channeling of carbamoyl phosphate a thermolabile intermediate evidence for physical interaction between carbamate kinase like carbamoyl phosphate synthetase and Ornithine Carbamoyltransferase from the hyperthermophile pyrococcus furiosus
Journal of Biological Chemistry, 2002Co-Authors: Jan Massant, Christianne Legrain, Patrik Verstreken, Virginie Durbecq, Abdelaziz Kholti, Sonia Beeckmans, Pierre Cornelis, Nicolas GlansdorffAbstract:Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and Ornithine Carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.
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Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase
Journal of Bacteriology, 2001Co-Authors: Martine Roovers, Christianne Legrain, Rony Sanchez, Nicolas GlansdorffAbstract:We have obtained mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15°C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.
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the crystal structure of pyrococcus furiosus Ornithine Carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Vincent Villeret, Christianne Legrain, Martine Roovers, Nicolas Glansdorff, Victor Stalon, Bernard Clantin, Catherine Tricot, Jozef Van BeeumenAbstract:The Pyrococcus furiosus (PF) Ornithine Carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100°C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 Å and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis–Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.
Christianne Legrain - One of the best experts on this subject based on the ideXlab platform.
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metabolic channeling of carbamoyl phosphate a thermolabile intermediate evidence for physical interaction between carbamate kinase like carbamoyl phosphate synthetase and Ornithine Carbamoyltransferase from the hyperthermophile pyrococcus furiosus
Journal of Biological Chemistry, 2002Co-Authors: Jan Massant, Christianne Legrain, Patrik Verstreken, Virginie Durbecq, Abdelaziz Kholti, Sonia Beeckmans, Pierre Cornelis, Nicolas GlansdorffAbstract:Two different approaches provided evidence for a physical interaction between the carbamate kinase-like carbamoyl-phosphate synthetase (CKase) and Ornithine Carbamoyltransferase (OTCase) from the hyperthermophilic archaeon Pyrococcus furiosus. Affinity electrophoresis indicated that CKase and OTCase associate into a multienzyme cluster. Further evidence for a biologically significant interaction between CKase and OTCase was obtained by co-immunoprecipitation combined with formaldehyde cross-linking experiments. These experiments support the hypothesis that CKase and OTCase form an efficient channeling cluster for carbamoyl phosphate, an extremely thermolabile and potentially toxic metabolic intermediate. Therefore, by physically interacting with each other, CKase and OTCase prevent the thermodenaturation of carbamoyl phosphate in the aqueous cytoplasmic environment.
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Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase
Journal of Bacteriology, 2001Co-Authors: Martine Roovers, Christianne Legrain, Rony Sanchez, Nicolas GlansdorffAbstract:We have obtained mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15°C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.
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the crystal structure of pyrococcus furiosus Ornithine Carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Vincent Villeret, Christianne Legrain, Martine Roovers, Nicolas Glansdorff, Victor Stalon, Bernard Clantin, Catherine Tricot, Jozef Van BeeumenAbstract:The Pyrococcus furiosus (PF) Ornithine Carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100°C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 Å and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis–Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.
Stalon Victor - One of the best experts on this subject based on the ideXlab platform.
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Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic Ornithine Carbamoyltransferase from Pseudomonas aeruginosa
2017Co-Authors: Seitz Thomas, Villeret Vincent, Tricot Catherine, Stalon Victor, Berger Bernard, Nguyen Van Thanh, Schmid Sergio, Haas DieterAbstract:The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic Ornithine Carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate Ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of protein
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Yeast epiarginase regulation, an enzyme-enzyme activity control: identification of residues of Ornithine Carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities.
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2003Co-Authors: El Alami Mohammed, Tricot Catherine, Stalon Victor, Dubois Evelyne, Oudjama Yamina, Wouters Johan, Messenguy FrancineAbstract:In the presence of Ornithine and arginine, Ornithine Carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative Ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for Ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to Ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by Ornithine for the closure of the catalytic domain.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe
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Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus Ornithine Carbamoyltransferase by site-specific mutants
'Wiley', 2001Co-Authors: Clantin Bernard, Stalon Victor, Tricot Catherine, Lonhienne Thierry, Villeret VincentAbstract:The Pyrococcus furiosus Ornithine Carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 degreesC. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization
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Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus Ornithine Carbamoyltransferase by site-specific mutants
'Wiley', 2001Co-Authors: Clantin Bernard, Stalon Victor, Tricot Catherine, Lonhienne Thierry, Villeret VincentAbstract:The Pyrococcus furiosus Ornithine Carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100°C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
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Linker insertion mutagenesis based on IS21 transposition: Isolation of an AMP-insensitive variant of catabolic Ornithine Carbamoyltransferase from Pseudomonas aeruginosa
2000Co-Authors: Seitz Thomas, Villeret Vincent, Nguyen Van, Tricot Catherine, Stalon Victor, Haas Didier, Berger Bernard, Schmid SergioAbstract:The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic Ornithine Carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate Ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
Martine Roovers - One of the best experts on this subject based on the ideXlab platform.
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Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase
Journal of Bacteriology, 2001Co-Authors: Martine Roovers, Christianne Legrain, Rony Sanchez, Nicolas GlansdorffAbstract:We have obtained mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15°C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.
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the crystal structure of pyrococcus furiosus Ornithine Carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Vincent Villeret, Christianne Legrain, Martine Roovers, Nicolas Glansdorff, Victor Stalon, Bernard Clantin, Catherine Tricot, Jozef Van BeeumenAbstract:The Pyrococcus furiosus (PF) Ornithine Carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100°C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 Å and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis–Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.
Tricot Catherine - One of the best experts on this subject based on the ideXlab platform.
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Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic Ornithine Carbamoyltransferase from Pseudomonas aeruginosa
2017Co-Authors: Seitz Thomas, Villeret Vincent, Tricot Catherine, Stalon Victor, Berger Bernard, Nguyen Van Thanh, Schmid Sergio, Haas DieterAbstract:The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic Ornithine Carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate Ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of protein
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Yeast epiarginase regulation, an enzyme-enzyme activity control: identification of residues of Ornithine Carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities.
'American Society for Biochemistry & Molecular Biology (ASBMB)', 2003Co-Authors: El Alami Mohammed, Tricot Catherine, Stalon Victor, Dubois Evelyne, Oudjama Yamina, Wouters Johan, Messenguy FrancineAbstract:In the presence of Ornithine and arginine, Ornithine Carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative Ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for Ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to Ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by Ornithine for the closure of the catalytic domain.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe
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Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus Ornithine Carbamoyltransferase by site-specific mutants
'Wiley', 2001Co-Authors: Clantin Bernard, Stalon Victor, Tricot Catherine, Lonhienne Thierry, Villeret VincentAbstract:The Pyrococcus furiosus Ornithine Carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 degreesC. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization
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Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus Ornithine Carbamoyltransferase by site-specific mutants
'Wiley', 2001Co-Authors: Clantin Bernard, Stalon Victor, Tricot Catherine, Lonhienne Thierry, Villeret VincentAbstract:The Pyrococcus furiosus Ornithine Carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100°C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.SCOPUS: ar.jinfo:eu-repo/semantics/publishe
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Linker insertion mutagenesis based on IS21 transposition: Isolation of an AMP-insensitive variant of catabolic Ornithine Carbamoyltransferase from Pseudomonas aeruginosa
2000Co-Authors: Seitz Thomas, Villeret Vincent, Nguyen Van, Tricot Catherine, Stalon Victor, Haas Didier, Berger Bernard, Schmid SergioAbstract:The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic Ornithine Carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate Ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.SCOPUS: ar.jinfo:eu-repo/semantics/publishe