Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius (459 views)
Proteins (ISSN: 0887-3585, 1097-0134, 1097-0134electronic), 2008 Jun; 71(4): 1721-1731.
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Paper type: Journal Article, Research Support, Non-U. S. Gov'T,
Impact factor: 3.419, 5-year impact factor: 4.019
Url: http://www.scopus.com/inward/record.url?eid=2-s2.0-44349174350&partnerID=40&md5=2f6dc5b338e70245557922223751d466
Keywords: Carboxylesterase, Oxyanion Loop, Ph-Activity Profile, Reaction Mechanism, Thermophilicity, 4 (2 Hydroxyethyl) 1 Piperazineethanesulfonic Acid, Mutant Protein, Alicyclobacillus Acidocaldarius, Article, Catalysis, Hydrogen Bond, Molecular Mechanics, Nonhuman, Priority Journal, Protein Function, Protein Structure, Temperature, Thermostability, Three Dimensional Imaging, Binding Sites, Carboxylic Ester Hydrolases, Crystallography, X-Ray, Enzyme Stability, Glycine, Hydrogen Bonding, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Models, Mutation, Protein Binding, Secondary, Serine, Substrate Specificity, Water, X-Ray Diffraction,
Affiliations:
*** IBB - CNR ***
Istituto di Biochimica Delle Proteine, CNR, Via Pietro Castellino 111, 80131 Naples, Italy
Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Naples, Italy
Bioindustry Park del Canavese, Via Ribes 5, 10010 Collerette Giacosa (To), Italy
Dipartimento Delle Scienze Biologiche, University of Naples Federico II, via Mezzocannone 16, 80134 Naples, Italy
References:
Not available.
Proteins (ISSN: 0887-3585, 1097-0134, 1097-0134electronic), 2008 Jun; 71(4): 1721-1731.
Export to BibTeX Export to EndNote
Paper type: Journal Article, Research Support, Non-U. S. Gov'T,
Impact factor: 3.419, 5-year impact factor: 4.019
Url: http://www.scopus.com/inward/record.url?eid=2-s2.0-44349174350&partnerID=40&md5=2f6dc5b338e70245557922223751d466
Keywords: Carboxylesterase, Oxyanion Loop, Ph-Activity Profile, Reaction Mechanism, Thermophilicity, 4 (2 Hydroxyethyl) 1 Piperazineethanesulfonic Acid, Mutant Protein, Alicyclobacillus Acidocaldarius, Article, Catalysis, Hydrogen Bond, Molecular Mechanics, Nonhuman, Priority Journal, Protein Function, Protein Structure, Temperature, Thermostability, Three Dimensional Imaging, Binding Sites, Carboxylic Ester Hydrolases, Crystallography, X-Ray, Enzyme Stability, Glycine, Hydrogen Bonding, Hydrogen-Ion Concentration, Hydrolysis, Kinetics, Models, Mutation, Protein Binding, Secondary, Serine, Substrate Specificity, Water, X-Ray Diffraction,
Affiliations:
*** IBB - CNR ***
Istituto di Biochimica Delle Proteine, CNR, Via Pietro Castellino 111, 80131 Naples, Italy
Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, 80134 Naples, Italy
Bioindustry Park del Canavese, Via Ribes 5, 10010 Collerette Giacosa (To), Italy
Dipartimento Delle Scienze Biologiche, University of Naples Federico II, via Mezzocannone 16, 80134 Naples, Italy
References:
Not available.
Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius
Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k cat were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65°C and pH = 5.75). The catalytic constants KM and V max were found considerably altered in the mutant, with ninefold increased KM and 14-fold decreased Vmax, at pH 5.75. At pH 7.1, the decrease in kcat was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate. © 2007 Wiley-Liss, Inc.
Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k cat were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65°C and pH = 5.75). The catalytic constants KM and V max were found considerably altered in the mutant, with ninefold increased KM and 14-fold decreased Vmax, at pH 5.75. At pH 7.1, the decrease in kcat was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate. © 2007 Wiley-Liss, Inc.
Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius
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Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius
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De Simone G, Menchise V, Alterio V, Mandrich L, Rossi M, Manco G, Pedone C
* The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase/lipase family (1052 views)
J Mol Biol (ISSN: 0022-2836, 1089-8638, 1089-8638electronic), 2004 Oct 8; 343(1): 137-146.
Impact Factor: 5.542
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De Simone G, Mandrich L, Menchise V, Giordano V, Febbraio F, Rossi M, Pedone C, Manco G
* A substrate-induced switch in the reaction mechanism of a thermophilic esterase - Kinetic evidences and structural basis (546 views)
Jbc Papers (ISSN: 0021-9258, 1083-351x, 0021-9258linking), 2004 Feb 20; 279(8): 6815-6823.
Impact Factor: 6.355
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De Simone G, Menchise V, Manco G, Mandrich L, Sorrentino N, Lang D, Rossi M, Pedone C
* The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus (610 views)
J Mol Biol (ISSN: 0022-2836, 1089-8638, 1089-8638electronic), 2001 Nov 30; 314(3): 507-518.
Impact Factor: 5.826
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De Simone G, Galdiero S, Manco G, Lang D, Rossi M, Pedone C
A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase (794 views)
J Mol Biol (ISSN: 0022-2836, 1089-8638, 1089-8638electronic), 2000 Nov 10; 303(5): 761-771.
Impact Factor: 5.388
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De Simone G, Manco G, Galdiero S, Lombardi A, Rossi M, Pavone V
Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius (479 views)
Acta Crystallogr D Biol Crystallogr (ISSN: 0907-4449, 0907-4449linking), 1999 Jul; 55(7): 1348-1349.
Impact Factor: 2.935
View Export to BibTeX Export to EndNote
* The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase/lipase family (1052 views)
J Mol Biol (ISSN: 0022-2836, 1089-8638, 1089-8638electronic), 2004 Oct 8; 343(1): 137-146.
Impact Factor: 5.542
View Export to BibTeX Export to EndNote
De Simone G, Mandrich L, Menchise V, Giordano V, Febbraio F, Rossi M, Pedone C, Manco G
* A substrate-induced switch in the reaction mechanism of a thermophilic esterase - Kinetic evidences and structural basis (546 views)
Jbc Papers (ISSN: 0021-9258, 1083-351x, 0021-9258linking), 2004 Feb 20; 279(8): 6815-6823.
Impact Factor: 6.355
View Export to BibTeX Export to EndNote
De Simone G, Menchise V, Manco G, Mandrich L, Sorrentino N, Lang D, Rossi M, Pedone C
* The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus (610 views)
J Mol Biol (ISSN: 0022-2836, 1089-8638, 1089-8638electronic), 2001 Nov 30; 314(3): 507-518.
Impact Factor: 5.826
View Export to BibTeX Export to EndNote
De Simone G, Galdiero S, Manco G, Lang D, Rossi M, Pedone C
A snapshot of a transition state analogue of a novel thermophilic esterase belonging to the subfamily of mammalian hormone-sensitive lipase (794 views)
J Mol Biol (ISSN: 0022-2836, 1089-8638, 1089-8638electronic), 2000 Nov 10; 303(5): 761-771.
Impact Factor: 5.388
View Export to BibTeX Export to EndNote
De Simone G, Manco G, Galdiero S, Lombardi A, Rossi M, Pavone V
Crystallization and preliminary X-ray diffraction studies of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius (479 views)
Acta Crystallogr D Biol Crystallogr (ISSN: 0907-4449, 0907-4449linking), 1999 Jul; 55(7): 1348-1349.
Impact Factor: 2.935
View Export to BibTeX Export to EndNote
5 Records (5 excluding Abstracts).
Total impact factor: 26.046 (26.046 excluding Abstracts).
Total 5 year impact factor: 17.821 (17.821 excluding Abstracts).
Total impact factor: 26.046 (26.046 excluding Abstracts).
Total 5 year impact factor: 17.821 (17.821 excluding Abstracts).
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