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[PMID]:28375146
[Au] Autor:Arif SM; Patil AG; Varshney U; Vijayan M
[Ad] Endereço:Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
[Ti] Título:Biochemical and structural studies of Mycobacterium smegmatis MutT1, a sanitization enzyme with unusual modes of association.
[So] Source:Acta Crystallogr D Struct Biol;73(Pt 4):349-364, 2017 Apr 01.
[Is] ISSN:2059-7983
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Mycobacterium smegmatis MutT1, which is made up of a Nudix domain (domain 1) and a histidine phosphatase domain (domain 2), efficiently hydrolyses 8-oxo-GTP and 8-oxo-dGTP to the corresponding nucleoside diphosphates and phosphate in the presence of magnesium ions. Domain 1 alone hydrolyses nucleoside triphosphates less efficiently. Under high concentrations and over long periods, the full-length enzyme as well as domain 1 catalyses the hydrolysis of the nucleoside triphosphates to the respective nucleoside monophosphates and pyrophosphate. The role of domain 2 appears to be limited to speeding up the reaction. Crystal structures of the apoenzyme and those of ligand-bound enzyme prepared in the presence of 8-oxo-GTP or 8-oxo-dGTP and different concentrations of magnesium were determined. In all of the structures except one, the molecules arrange themselves in a head-to-tail fashion in which domain 1 is brought into contact with domain 2 (trans domain 2) of a neighbouring molecule. The binding site for NTP (site A) is almost exclusively made up of residues from domain 1, while those for NDP (site B) and NMP (site C) are at the interface between domain 1 and trans domain 2 in an unusual instance of intermolecular interactions leading to binding sites. Protein-ligand interactions at site A lead to a proposal for the mechanism of hydrolysis of NTP to NDP and phosphate. A small modification in site A in the crystal which does not exhibit the head-to-tail arrangement appears to facilitate the production of NMP and pyrophosphate from NTP. The two arrangements could be in dynamic equilibrium in the cellular milieu.
[Mh] Termos MeSH primário: Proteínas de Bactérias/química
Mycobacterium smegmatis/química
Hidrolases de Triester Fosfórico/química
[Mh] Termos MeSH secundário: Proteínas de Bactérias/metabolismo
Cristalografia por Raios X
Nucleotídeos de Desoxiguanina/metabolismo
Guanosina Trifosfato/análogos & derivados
Guanosina Trifosfato/metabolismo
Seres Humanos
Modelos Moleculares
Infecções por Micobactéria não Tuberculosa/microbiologia
Mycobacterium smegmatis/metabolismo
Hidrolases de Triester Fosfórico/metabolismo
Conformação Proteica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Deoxyguanine Nucleotides); 139307-94-1 (8-oxodeoxyguanosine triphosphate); 21238-36-8 (8-hydroxyguanosine triphosphate); 86-01-1 (Guanosine Triphosphate); EC 3.1.8.- (Phosphoric Triester Hydrolases)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:171106
[Lr] Data última revisão:
171106
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170405
[St] Status:MEDLINE
[do] DOI:10.1107/S2059798317002534


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[PMID]:28323400
[Au] Autor:Hoque MA; Zhang Y; Chen L; Yang G; Khatun MA; Chen H; Hao L; Feng Y
[Ad] Endereço:State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
[Ti] Título:Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.
[So] Source:ACS Chem Biol;12(5):1188-1193, 2017 May 19.
[Is] ISSN:1554-8937
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.
[Mh] Termos MeSH primário: Proteínas de Bactérias/genética
Hidrolases de Éster Carboxílico/genética
Domínio Catalítico/genética
Geobacillus/enzimologia
Mutagênese Insercional/métodos
Engenharia de Proteínas/métodos
[Mh] Termos MeSH secundário: Proteínas de Bactérias/metabolismo
Biocatálise
Hidrolases de Éster Carboxílico/metabolismo
Simulação de Acoplamento Molecular
Estrutura Molecular
Organofosfatos/metabolismo
Hidrolases de Triester Fosfórico/metabolismo
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Organophosphates); EC 3.1.1.- (Carboxylic Ester Hydrolases); EC 3.1.8.- (Phosphoric Triester Hydrolases)
[Em] Mês de entrada:1709
[Cu] Atualização por classe:170912
[Lr] Data última revisão:
170912
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170322
[St] Status:MEDLINE
[do] DOI:10.1021/acschembio.7b00018


  3 / 212 MEDLINE  
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[PMID]:26775655
[Au] Autor:Guan S; Zhao L; Jin H; Shan N; Han W; Wang S; Shan Y
[Ad] Endereço:a National Engineering Laboratory For AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences , Jilin University , Changchun 130012 , China.
[Ti] Título:Binding modes of phosphotriesterase-like lactonase complexed with δ-nonanoic lactone and paraoxon using molecular dynamics simulations.
[So] Source:J Biomol Struct Dyn;35(2):273-286, 2017 Feb.
[Is] ISSN:1538-0254
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Phosphotriesterase-like lactonases (PLLs) have received much attention because of their physical and chemical properties. They may have widespread applications in various fields. For example, they show potential for quorum-sensing signaling pathways and organophosphorus (OP) detoxification in agricultural science. However, the mechanism by which PLLs hydrolyze, which involves OP compounds and lactones and a variety of distinct catalytic efficiencies, has only rarely been explored. In the present study, molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of DrPLL, a member of the PLL superfamily in Deinococcus radiodurans, bound to two substrates, δ-nonanoic lactone and paraoxon. It has been observed that there is a 16-fold increase in the catalytic efficiency of the two mutant strains of DrPLL (F26G/C72I) vs. the wild-type enzyme toward the hydrolysis of paraoxon, but an explanation for this behavior is currently lacking. The analysis of the molecular trajectories of DrPLL bound to δ-nonanoic lactone indicated that lactone-induced conformational changes take place in loop 8, which is near the active site. Binding to paraoxon may lead to conformational displacement of loop 1 residues, which could lead to the deformation of the active site and so trigger the entry of the paraoxon into the active site. The efficiency of the F26G/C72I mutant was increased by decreasing the displacement of loop 1 residues and increasing the flexibility of loop 8 residues. These results provide a molecular-level explanation for the experimental behavior.
[Mh] Termos MeSH primário: Lactonas/química
Simulação de Dinâmica Molecular
Paraoxon/química
Hidrolases de Triester Fosfórico/química
[Mh] Termos MeSH secundário: Sítios de Ligação
Ligações de Hidrogênio
Ligantes
Mutação
Hidrolases de Triester Fosfórico/genética
Ligação Proteica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Lactones); 0 (Ligands); EC 3.1.8.- (Phosphoric Triester Hydrolases); Q9CX8P80JW (Paraoxon)
[Em] Mês de entrada:1703
[Cu] Atualização por classe:170817
[Lr] Data última revisão:
170817
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160119
[St] Status:MEDLINE
[do] DOI:10.1080/07391102.2016.1142899


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[PMID]:27915249
[Au] Autor:Santillan JY; Dettorre LA; Lewkowicz ES; Iribarren AM
[Ad] Endereço:Laboratorio de Biocatálisis y Biotransformaciones, Universidad Nacional de Quilmes, 1876 Bernal, Buenos Aires, Argentina.
[Ti] Título:New and highly active microbial phosphotriesterase sources.
[So] Source:FEMS Microbiol Lett;363(24), 2016 Dec.
[Is] ISSN:1574-6968
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Many toxic insecticides used worldwide as well as some chemical warfare agents are phosphotriester derivatives. Therefore, detoxification of organophosphorus compounds has become the subject of many studies and in particular bioremediation, based on the phosphotriesterase catalysed hydrolysis of these compounds, has shown to be an effective and ecological methodology. In order to identify new bacterial phosphotriesterases, a simple and sensitive fluorimetric screening method on solid media was employed that allowed the selection of six strains with phosphotriesterase activity. Since pH and temperature are important parameters for bioremediation of contaminated soils and waters, the influence of these variables on the rate of the enzymatic hydrolysis was assessed. This study afforded notable results, being the most remarkable one the increased activity exhibited by Nocardia asteroides and Streptomyces setonii strains at 50°C, 7 and 30 times higher than at 30°C, respectively. Compared with the results obtained with Brevundimonas diminuta, whose activity is usually considered as reference, an increase of 26 and 75 times is observed, respectively.
[Mh] Termos MeSH primário: Caulobacteraceae/enzimologia
Hidrolases de Triester Fosfórico/análise
Streptomyces/enzimologia
[Mh] Termos MeSH secundário: Técnicas Bacteriológicas/métodos
Estabilidade Enzimática
Concentração de Íons de Hidrogênio
Programas de Rastreamento/métodos
Nocardia asteroides/enzimologia
Hidrolases de Triester Fosfórico/química
Temperatura Ambiente
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 3.1.8.- (Phosphoric Triester Hydrolases)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:170829
[Lr] Data última revisão:
170829
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161205
[St] Status:MEDLINE


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[PMID]:27716796
[Au] Autor:Kaltenbach M; Emond S; Hollfelder F; Tokuriki N
[Ad] Endereço:Michael Smith Laboratories, University of British Columbia, Vancouver, Canada.
[Ti] Título:Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.
[So] Source:PLoS Genet;12(10):e1006305, 2016 Oct.
[Is] ISSN:1553-7404
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1) from phosphotriesterase to arylesterase, and (2) from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak trade-offs and the emergence of generalist enzymes.
[Mh] Termos MeSH primário: Enzimas/genética
Evolução Molecular
[Mh] Termos MeSH secundário: Substituição de Aminoácidos/genética
Aminoidrolases/química
Aminoidrolases/genética
Hidrolases de Éster Carboxílico/química
Hidrolases de Éster Carboxílico/genética
Enzimas/química
Aptidão Genética
Hidrolases/química
Hidrolases/genética
Modelos Genéticos
Hidrolases de Triester Fosfórico/química
Hidrolases de Triester Fosfórico/genética
Mutação Puntual
Seleção Genética
Deleção de Sequência
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Enzymes); EC 3.- (Hydrolases); EC 3.1.1.- (Carboxylic Ester Hydrolases); EC 3.1.1.2 (arylesterase); EC 3.1.8.- (Phosphoric Triester Hydrolases); EC 3.5.4.- (Aminohydrolases); EC 3.5.4.- (melamine deaminase); EC 3.8.1.8 (atrazine chlorohydrolase)
[Em] Mês de entrada:1704
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161008
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pgen.1006305


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[PMID]:27618189
[Au] Autor:Campbell E; Kaltenbach M; Correy GJ; Carr PD; Porebski BT; Livingstone EK; Afriat-Jurnou L; Buckle AM; Weik M; Hollfelder F; Tokuriki N; Jackson CJ
[Ad] Endereço:Research School of Chemistry, Australian National University, Canberra, Australia.
[Ti] Título:The role of protein dynamics in the evolution of new enzyme function.
[So] Source:Nat Chem Biol;12(11):944-950, 2016 Nov.
[Is] ISSN:1552-4469
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
[Mh] Termos MeSH primário: Hidrolases de Éster Carboxílico/metabolismo
Evolução Molecular
Hidrolases de Triester Fosfórico/metabolismo
Pseudomonas/enzimologia
[Mh] Termos MeSH secundário: Biocatálise
Hidrolases de Éster Carboxílico/química
Hidrolases de Triester Fosfórico/química
Conformação Proteica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
EC 3.1.1.- (Carboxylic Ester Hydrolases); EC 3.1.1.2 (arylesterase); EC 3.1.8.- (Phosphoric Triester Hydrolases)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170606
[Lr] Data última revisão:
170606
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160913
[St] Status:MEDLINE
[do] DOI:10.1038/nchembio.2175


  7 / 212 MEDLINE  
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[PMID]:27444875
[Au] Autor:Yang G; Hong N; Baier F; Jackson CJ; Tokuriki N
[Ad] Endereço:Michael Smith Laboratories, University of British Columbia , Vancouver, BC V6T 1Z4, Canada.
[Ti] Título:Conformational Tinkering Drives Evolution of a Promiscuous Activity through Indirect Mutational Effects.
[So] Source:Biochemistry;55(32):4583-93, 2016 Aug 16.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:How remote mutations can lead to changes in enzyme function at a molecular level is a central question in evolutionary biochemistry and biophysics. Here, we combine laboratory evolution with biochemical, structural, genetic, and computational analysis to dissect the molecular basis for the functional optimization of phosphotriesterase activity in a bacterial lactonase (AiiA) from the metallo-ß-lactamase (MBL) superfamily. We show that a 1000-fold increase in phosphotriesterase activity is caused by a more favorable catalytic binding position of the paraoxon substrate in the evolved enzyme that resulted from conformational tinkering of the active site through peripheral mutations. A nonmutated active site residue, Phe68, was displaced by ∼3 Å through the indirect effects of two second-shell trajectory mutations, allowing molecular interactions between the residue and paraoxon. Comparative mutational scanning, i.e., examining the effects of alanine mutagenesis on different genetic backgrounds, revealed significant changes in the functional roles of Phe68 and other nonmutated active site residues caused by the indirect effects of trajectory mutations. Our work provides a quantitative measurement of the impact of second-shell mutations on the catalytic contributions of nonmutated residues and unveils the underlying intramolecular network of strong epistatic mutational relationships between active site residues and more remote residues. Defining these long-range conformational and functional epistatic relationships has allowed us to better understand the subtle, but cumulatively significant, role of second- and third-shell mutations in evolution.
[Mh] Termos MeSH primário: Proteínas de Bactérias/química
Proteínas de Bactérias/metabolismo
Evolução Molecular Direcionada
Metaloendopeptidases/química
Metaloendopeptidases/metabolismo
Mutação
[Mh] Termos MeSH secundário: Proteínas de Bactérias/genética
Biocatálise
Domínio Catalítico
Genótipo
Metaloendopeptidases/genética
Simulação de Acoplamento Molecular
Simulação de Dinâmica Molecular
Hidrolases de Triester Fosfórico/metabolismo
Especificidade por Substrato
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Bacterial Proteins); EC 3.1.8.- (Phosphoric Triester Hydrolases); EC 3.4.24.- (AiiA protein, Bacillus); EC 3.4.24.- (Metalloendopeptidases)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170501
[Lr] Data última revisão:
170501
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160723
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b00561


  8 / 212 MEDLINE  
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[PMID]:27425410
[Au] Autor:Goldenzweig A; Goldsmith M; Hill SE; Gertman O; Laurino P; Ashani Y; Dym O; Unger T; Albeck S; Prilusky J; Lieberman RL; Aharoni A; Silman I; Sussman JL; Tawfik DS; Fleishman SJ
[Ad] Endereço:Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 7610001, Israel.
[Ti] Título:Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.
[So] Source:Mol Cell;63(2):337-346, 2016 Jul 21.
[Is] ISSN:1097-4164
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
[Mh] Termos MeSH primário: Acetilcolinesterase/metabolismo
Biologia Computacional/métodos
Escherichia coli/enzimologia
Engenharia de Proteínas/métodos
[Mh] Termos MeSH secundário: Acetilcolinesterase/química
Acetilcolinesterase/genética
Algoritmos
Automação Laboratorial
Simulação por Computador
Projeto Auxiliado por Computador
DNA (Citosina-5-)-Metiltransferases/genética
DNA (Citosina-5-)-Metiltransferases/metabolismo
Escherichia coli/genética
Proteínas Ligadas por GPI/química
Proteínas Ligadas por GPI/genética
Proteínas Ligadas por GPI/metabolismo
Regulação Bacteriana da Expressão Gênica
Regulação Enzimológica da Expressão Gênica
Mutação
Hidrolases de Triester Fosfórico/genética
Hidrolases de Triester Fosfórico/metabolismo
Conformação Proteica
Desnaturação Proteica
Estabilidade Proteica
Sirtuínas/genética
Sirtuínas/metabolismo
Relação Estrutura-Atividade
Temperatura Ambiente
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (GPI-Linked Proteins); EC 2.1.1.37 (DNA (Cytosine-5-)-Methyltransferases); EC 2.1.1.37 (DNA methyltransferase 3A); EC 3.1.1.7 (ACHE protein, human); EC 3.1.1.7 (Acetylcholinesterase); EC 3.1.8.- (Phosphoric Triester Hydrolases); EC 3.5.1.- (SIRT6 protein, human); EC 3.5.1.- (Sirtuins)
[Em] Mês de entrada:1708
[Cu] Atualização por classe:171116
[Lr] Data última revisão:
171116
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160719
[St] Status:MEDLINE


  9 / 212 MEDLINE  
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[PMID]:27397758
[Au] Autor:Wille T; Neumaier K; Koller M; Ehinger C; Aggarwal N; Ashani Y; Goldsmith M; Sussman JL; Tawfik DS; Thiermann H; Worek F
[Ad] Endereço:Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany. Electronic address: timowille@bundeswehr.org.
[Ti] Título:Single treatment of VX poisoned guinea pigs with the phosphotriesterase mutant C23AL: Intraosseous versus intravenous injection.
[So] Source:Toxicol Lett;258:198-206, 2016 Sep 06.
[Is] ISSN:1879-3169
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The recent attacks with the nerve agent sarin in Syria reveal the necessity of effective countermeasures against highly toxic organophosphorus compounds. Multiple studies provide evidence that a rapid onset of antidotal therapy might be life-saving but current standard antidotal protocols comprising reactivators and competitive muscarinic antagonists show a limited efficacy for several nerve agents. We here set out to test the newly developed phosphotriesterase (PTE) mutant C23AL by intravenous (i.v.), intramuscular (i.m.; model for autoinjector) and intraosseous (i.o.; model for intraosseous insertion device) application in an in vivo guinea pig model after VX challenge (∼2LD50). C23AL showed a Cmax of 0.63µmolL(-1) after i.o. and i.v. administration of 2mgkg(-1) providing a stable plasma profile up to 180min experimental duration with 0.41 and 0.37µmolL(-1) respectively. The i.m. application of C23AL did not result in detectable plasma levels. All animals challenged with VX and subsequent i.o. or i.v. C23AL therapy survived although an in part substantial inhibition of erythrocyte, brain and diaphragm AChE was detected. Theoretical calculation of the time required to hydrolyze in vivo 96.75% of the toxic VX enantiomer is consistent with previous studies wherein similar activity of plasma containing catalytic scavengers of OPs resulted in non-lethal protection although accompanied with a variable severity of cholinergic symptoms. The relatively low C23AL plasma level observed immediately after its i.v. or i.o load, point at a possible volume of distribution greater than the guinea pig plasma content, and thus underlines the necessity of in vivo experiments in antidote research. In conclusion the i.o. application of PTE is efficient and resulted in comparable plasma levels to the i.v. application at a given time. Thus, i.o. vascular access systems could improve the post-exposure PTE therapy of nerve agent poisoning.
[Mh] Termos MeSH primário: Antídotos/administração & dosagem
Mutação
Agentes Neurotóxicos/toxicidade
Intoxicação por Organofosfatos/tratamento farmacológico
Compostos Organotiofosforados/toxicidade
Fragmentos de Peptídeos/administração & dosagem
Hidrolases de Triester Fosfórico/administração & dosagem
[Mh] Termos MeSH secundário: Animais
Animais não Endogâmicos
Antídotos/metabolismo
Antídotos/farmacocinética
Antídotos/uso terapêutico
Proteínas de Bactérias/administração & dosagem
Proteínas de Bactérias/genética
Proteínas de Bactérias/farmacocinética
Proteínas de Bactérias/uso terapêutico
Medula Óssea
Cobaias
Inativação Metabólica
Injeções Intralesionais
Injeções Intramusculares
Injeções Intravenosas
Injeções Subcutâneas
Masculino
Agentes Neurotóxicos/análise
Agentes Neurotóxicos/metabolismo
Intoxicação por Organofosfatos/sangue
Intoxicação por Organofosfatos/etiologia
Intoxicação por Organofosfatos/metabolismo
Compostos Organotiofosforados/administração & dosagem
Compostos Organotiofosforados/antagonistas & inibidores
Compostos Organotiofosforados/metabolismo
Fragmentos de Peptídeos/metabolismo
Fragmentos de Peptídeos/farmacocinética
Fragmentos de Peptídeos/uso terapêutico
Hidrolases de Triester Fosfórico/genética
Hidrolases de Triester Fosfórico/farmacocinética
Hidrolases de Triester Fosfórico/uso terapêutico
Proteólise
Pseudomonas/enzimologia
Proteínas Recombinantes/administração & dosagem
Proteínas Recombinantes/metabolismo
Proteínas Recombinantes/farmacocinética
Proteínas Recombinantes/uso terapêutico
Toxicocinética
[Pt] Tipo de publicação:COMPARATIVE STUDY; JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Antidotes); 0 (Bacterial Proteins); 0 (Nerve Agents); 0 (Organothiophosphorus Compounds); 0 (Peptide Fragments); 0 (Recombinant Proteins); 9A4381183B (VX); EC 3.1.8.- (Phosphoric Triester Hydrolases)
[Em] Mês de entrada:1702
[Cu] Atualização por classe:170926
[Lr] Data última revisão:
170926
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160712
[St] Status:MEDLINE


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[PMID]:27353520
[Au] Autor:Mabanglo MF; Xiang DF; Bigley AN; Raushel FM
[Ad] Endereço:Department of Chemistry, Texas A&M University , P.O. Box 30012, College Station, Texas 77842, United States.
[Ti] Título:Structure of a Novel Phosphotriesterase from Sphingobium sp. TCM1: A Familiar Binuclear Metal Center Embedded in a Seven-Bladed ß-Propeller Protein Fold.
[So] Source:Biochemistry;55(28):3963-74, 2016 Jul 19.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:A novel phosphotriesterase was recently discovered and purified from Sphingobium sp. TCM1 (Sb-PTE) and shown to catalyze the hydrolysis of a broad spectrum of organophosphate esters with a catalytic efficiency that exceeds 10(6) M(-1) s(-1) for the hydrolysis of triphenyl phosphate. The enzyme was crystallized and the three-dimensional structure determined to a resolution of 2.1 Å using single-wavelength anomalous diffraction (Protein Data Bank entry 5HRM ). The enzyme adopts a seven-bladed ß-propeller protein fold, and three disulfide bonds were identified between Cys-146 and Cys-242, Cys-411 and Cys-443, and Cys-542 and Cys-559. The active site of Sb-PTE contains a binuclear manganese center that is nearly identical to that of the structurally unrelated phosphotriesterase from Pseudomonas diminuta (Pd-PTE). The two metal ions in the active site are bridged to one another by Glu-201 and a water molecule. The α-metal ion is further coordinated to the protein by interactions with His-389, His-475, and Glu-407, whereas the ß-metal ion is further liganded to His-317 and His-258. Computational docking of mimics of the proposed pentavalent reaction intermediates for the hydrolysis of organophosphates was used to provide a model for the binding of chiral substrates in the active site of Sb-PTE. The most striking difference in the catalytic properties of Sb-PTE, relative to those of Pd-PTE, is the enhanced rate of hydrolysis of organophosphate esters with substantially weaker leaving groups. The structural basis for this difference in the catalytic properties between Sb-PTE and Pd-PTE, despite the nearly identical binuclear metal centers for the activation of the substrate and nucleophilic water molecule, is at present unclear.
[Mh] Termos MeSH primário: Manganês
Hidrolases de Triester Fosfórico/química
Hidrolases de Triester Fosfórico/metabolismo
Sphingomonadaceae/enzimologia
[Mh] Termos MeSH secundário: Biocatálise
Domínio Catalítico
Cristalografia por Raios X
Simulação de Acoplamento Molecular
Mutagênese
Hidrolases de Triester Fosfórico/genética
Conformação Proteica em Folha beta
Multimerização Proteica
Estrutura Quaternária de Proteína
Subunidades Proteicas/química
Subunidades Proteicas/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Protein Subunits); 42Z2K6ZL8P (Manganese); EC 3.1.8.- (Phosphoric Triester Hydrolases)
[Em] Mês de entrada:1705
[Cu] Atualização por classe:170501
[Lr] Data última revisão:
170501
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:160630
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.6b00364



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