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[PMID]:28548844
[Au] Autor:Kohne M; Zhu C; Warncke K
[Ad] Endereço:Department of Physics, Emory University , Atlanta, Georgia 30322, United States.
[Ti] Título:Two Dynamical Regimes of the Substrate Radical Rearrangement Reaction in B -Dependent Ethanolamine Ammonia-Lyase Resolve Contributions of Native Protein Configurations and Collective Configurational Fluctuations to Catalysis.
[So] Source:Biochemistry;56(25):3257-3264, 2017 Jun 27.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:The kinetics of the substrate radical rearrangement reaction step in B -dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium are measured over a 92 K temperature range. The observed first-order rate constants display a piecewise-continuous Arrhenius dependence, with linear regions over 295 → 220 K (monoexponential) and 214 → 203 K (biexponential) that are delineated by a kinetic bifurcation and kinks at 219 and 217 K, respectively. The results are interpreted by using a free energy landscape model and derived microscopic kinetic mechanism. The bifurcation and kink transitions correspond to the effective quenching of two distinct sets of native collective protein configurational fluctuations that (1) reconfigure the protein within the substrate radical free energy minimum, in a reaction-enabling step, and (2) create the protein configurations associated with the chemical step. Below 217 K, the substrate radical decay reaction persists. Increases in activation enthalpy and entropy of both the microscopic enabling and reaction steps indicate that this non-native reaction coordinate is conducted by local, incremental fluctuations. Continuity in the Arrhenius relations indicates that the same sets of protein groups and interactions mediate the rearrangement over the 295 to 203 K range, but with a repertoire of configurations below 217 K that is restricted, relative to the native configurations accessible above 219 K. The experimental features of a culled reaction step, first-order kinetic measurements, and wide room-to-cryogenic temperature range, allow the direct demonstration and kinetic characterization of protein dynamical contributions to the core adiabatic, bond-making/bond-breaking reaction in EAL.
[Mh] Termos MeSH primário: Cobalto/química
Cobamidas/química
Etanolamina Amônia-Liase/química
Salmonella typhimurium/enzimologia
[Mh] Termos MeSH secundário: Catálise
Cobalto/metabolismo
Cobamidas/metabolismo
Espectroscopia de Ressonância de Spin Eletrônica
Etanolamina Amônia-Liase/metabolismo
Radicais Livres
Cinética
Termodinâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (Cobamides); 0 (Free Radicals); 3G0H8C9362 (Cobalt); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase)
[Em] Mês de entrada:1707
[Cu] Atualização por classe:170907
[Lr] Data última revisão:
170907
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170527
[St] Status:MEDLINE
[do] DOI:10.1021/acs.biochem.7b00294


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[PMID]:26478482
[Au] Autor:Wang M; Zhu C; Kohne M; Warncke K
[Ad] Endereço:Department of Physics, Emory University, N201 Mathematics and Science Center, Atlanta, Georgia, USA.
[Ti] Título:Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems.
[So] Source:Methods Enzymol;563:59-94, 2015.
[Is] ISSN:1557-7988
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Approaches to the resolution and characterization of individual chemical steps in enzyme catalytic sequences, by using temperatures in the cryogenic range of 190-250 K, and kinetics measured by time-resolved, full-spectrum electron paramagnetic resonance spectroscopy in fluid cryosolvent and frozen solution systems, are described. The preparation and performance of the adenosylcobalamin-dependent ethanolamine ammonia-lyase enzyme from Salmonella typhimurium in the two systems exemplifies the biochemical and spectroscopic methods. General advantages of low-temperature studies are (1) slowing of reaction steps, so that measurements can be made by using straightforward T-step kinetic methods and commercial instrumentation, (2) resolution of individual reaction steps, so that first-order kinetic analysis can be applied, and (3) accumulation of intermediates that are not detectable at room temperatures. The broad temperature range from room temperature to 190 K encompasses three regimes: (1) temperature-independent mean free energy surface (corresponding to native behavior); (2) the narrow temperature region of a glass-like transition in the protein, over which the free energy surface changes, revealing dependence of the native reaction on collective protein/solvent motions; and (3) the temperature range below the glass transition region, for which persistent reaction corresponds to nonnative, alternative reaction pathways, in the vicinity of the native configurational envelope. Representative outcomes of low-temperature kinetics studies are portrayed on Eyring and free energy surface (landscape) plots, and guidelines for interpretations are presented.
[Mh] Termos MeSH primário: Espectroscopia de Ressonância de Spin Eletrônica/métodos
Etanolamina Amônia-Liase/química
Salmonella typhimurium/enzimologia
[Mh] Termos MeSH secundário: Catálise
Cobamidas/metabolismo
Temperatura Baixa
Etanolamina Amônia-Liase/metabolismo
Cinética
Conformação Proteica
Salmonella typhimurium/química
Solventes/química
Vitamina B 12/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cobamides); 0 (Solvents); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase); F0R1QK73KB (cobamamide); P6YC3EG204 (Vitamin B 12)
[Em] Mês de entrada:1607
[Cu] Atualização por classe:161125
[Lr] Data última revisão:
161125
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:151020
[St] Status:MEDLINE


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[PMID]:25950663
[Au] Autor:Jones AR; Rentergent J; Scrutton NS; Hay S
[Ad] Endereço:School of Chemistry, Manchester Institute of Biotechnology and Photon Science Institute, The University of Manchester, Alan Turing Building, Oxford Road, Manchester M13 9PL (UK). alex.jones-2@manchester.ac.uk.
[Ti] Título:Probing reversible chemistry in coenzyme B12 -dependent ethanolamine ammonia lyase with kinetic isotope effects.
[So] Source:Chemistry;21(24):8826-31, 2015 Jun 08.
[Is] ISSN:1521-3765
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Coenzyme B12 -dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the ß-carbon of the 5'-deoxyadenosyl moiety of the intrinsic coenzyme B12 , it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5'-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5'-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small.
[Mh] Termos MeSH primário: Cobamidas/química
Etanolamina Amônia-Liase/química
[Mh] Termos MeSH secundário: Catálise
Cinética
Modelos Moleculares
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cobamides); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase)
[Em] Mês de entrada:1603
[Cu] Atualização por classe:161019
[Lr] Data última revisão:
161019
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:150508
[St] Status:MEDLINE
[do] DOI:10.1002/chem.201500958


  4 / 100 MEDLINE  
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[PMID]:24735254
[Au] Autor:Mori K; Oiwa T; Kawaguchi S; Kondo K; Takahashi Y; Toraya T
[Ad] Endereço:Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University , Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
[Ti] Título:Catalytic roles of substrate-binding residues in coenzyme B12-dependent ethanolamine ammonia-lyase.
[So] Source:Biochemistry;53(16):2661-71, 2014 Apr 29.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Ethanolamine ammonia-lyase (EAL) catalyzes the adenosylcobalamin-dependent conversion of ethanolamine to acetaldehyde and ammonia. 1-OH of the substrate is hydrogen-bonded with Gluα287, Argα160, and Asnα193 and 2-NH2 with Gluα287, Glnα162, and Aspα362. The active site somewhat resembles that of diol dehydratase. All five residues were important for the high-affinity binding of the substrate and for catalysis. The -COO(-) group at residue α287 was absolutely required for activity and coenzyme Co-C bond cleavage, and there was a spatially optimal position for it, suggesting that Gluα287 contributes to Co-C bond homolysis, stabilizes the transition state for the migration of NH2 from C2 to C1 through partial deprotonation of spectator OH, and functions as a base in the elimination of ammonia. A positive charge and/or the hydrogen bond at position α160 and the hydrogen bonds at positions α162 and α193 with the substrate are important for catalysis and for preventing a radical intermediate from undergoing side reactions. Argα160 would stabilize the trigonal transition state in NH2 migration by electrostatic catalysis and hydrogen bonding with spectator OH. Asnα193 would contribute to maintaining the appropriate position and direction of the guanidinium group of Argα160, as well. Hydrogen bond acceptors were necessary at position α162, but hydrogen bond donors were rather harmful. Glnα162 might stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+). The activity was very sensitive to the position of -COO(-) at α362. Aspα362 would assist Co-C bond homolysis indirectly and stabilize the trigonal transition state by accepting a hydrogen bond from migrating NH3(+) and electrostatic interaction.
[Mh] Termos MeSH primário: Etanolamina Amônia-Liase/química
Etanolamina Amônia-Liase/metabolismo
[Mh] Termos MeSH secundário: Sítios de Ligação
Catálise
Domínio Catalítico
Cobamidas/metabolismo
Espectroscopia de Ressonância de Spin Eletrônica
Proteínas de Escherichia coli/química
Proteínas de Escherichia coli/genética
Proteínas de Escherichia coli/metabolismo
Etanolamina Amônia-Liase/genética
Ligações de Hidrogênio
Mutagênese Sítio-Dirigida
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cobamides); 0 (Escherichia coli Proteins); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase); F0R1QK73KB (cobamamide)
[Em] Mês de entrada:1408
[Cu] Atualização por classe:161125
[Lr] Data última revisão:
161125
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:140417
[St] Status:MEDLINE
[do] DOI:10.1021/bi500223k


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[PMID]:24269950
[Au] Autor:Toraya T
[Ad] Endereço:Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Kita-ku, Japan. Electronic address: toraya@cc.okayama-u.ac.jp.
[Ti] Título:Cobalamin-dependent dehydratases and a deaminase: radical catalysis and reactivating chaperones.
[So] Source:Arch Biochem Biophys;544:40-57, 2014 Feb 15.
[Is] ISSN:1096-0384
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Adenosylcobalamin, a coenzyme form of vitamin B12, is an organometallic compound that participates in about ten enzymatic reactions. These enzymes catalyze chemically challenging reactions by using a highly reactive primary carbon radical that is derived from homolysis of the coenzyme Co-C bond. Among them, diol dehydratases and ethanolamine ammonia-lyase have been most extensively studied to establish the general mechanism of adenosylcobalamin-assisted enzymatic catalysis and radical-catalyzed reactions. Another important point is that adenosylcobalamin-dependent radical enzymes are prone to mechanism-based irreversible inactivation during catalysis and have their own chaperones for the maintenance of catalytic activities. This review will highlight biochemical, structural, and computational studies with special emphases on radical catalysis and reactivating chaperones of these enzymes.
[Mh] Termos MeSH primário: Etanolamina Amônia-Liase/metabolismo
Hidroliases/metabolismo
Vitamina B 12/metabolismo
[Mh] Termos MeSH secundário: Animais
Bactérias/química
Bactérias/enzimologia
Cristalografia por Raios X
Etanolamina Amônia-Liase/química
Seres Humanos
Hidroliases/química
Modelos Moleculares
Vitamina B 12/química
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T; REVIEW
[Nm] Nome de substância:
EC 4.2.1.- (Hydro-Lyases); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase); P6YC3EG204 (Vitamin B 12)
[Em] Mês de entrada:1404
[Cu] Atualização por classe:140204
[Lr] Data última revisão:
140204
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:131126
[St] Status:MEDLINE


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[PMID]:24028405
[Au] Autor:Wang M; Warncke K
[Ad] Endereço:Department of Physics, Emory University , Atlanta, Georgia 30322, United States.
[Ti] Título:Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase.
[So] Source:J Am Chem Soc;135(40):15077-84, 2013 Oct 09.
[Is] ISSN:1520-5126
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt-carbon (Co-C) bond of the bound coenzyme by >10(10)-fold. The cleavage-generated 5'-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co-C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co-C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme-coenzyme-substrate ternary complex and (2)H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from Salmonella typhimurium at 234-248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the (2)H- and (1)H-substituted substrate radicals are the same, indicating that Co-C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co-C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co-C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co-C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co-C bond-breaking in B12 enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate.
[Mh] Termos MeSH primário: Biocatálise
Carbono/química
Cobalto/química
Cobamidas/metabolismo
Entropia
Etanolamina Amônia-Liase/metabolismo
[Mh] Termos MeSH secundário: Etanolamina Amônia-Liase/química
Cinética
Modelos Moleculares
Propanolaminas/metabolismo
Conformação Proteica
Salmonella typhimurium/enzimologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cobamides); 0 (Propanolamines); 3G0H8C9362 (Cobalt); 7440-44-0 (Carbon); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase); F0R1QK73KB (cobamamide)
[Em] Mês de entrada:1405
[Cu] Atualização por classe:161125
[Lr] Data última revisão:
161125
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130914
[St] Status:MEDLINE
[do] DOI:10.1021/ja404467d


  7 / 100 MEDLINE  
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[PMID]:23959797
[Au] Autor:Chen ZG; Zietek MA; Russell HJ; Tait S; Hay S; Jones AR; Scrutton NS
[Ad] Endereço:College of Food and Science Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing 210095 (P.R. China).
[Ti] Título:Dynamic, electrostatic model for the generation and control of high-energy radical intermediates by a coenzyme B12-dependent enzyme.
[So] Source:Chembiochem;14(13):1529-33, 2013 Sep 02.
[Is] ISSN:1439-7633
[Cp] País de publicação:Germany
[La] Idioma:eng
[Mh] Termos MeSH primário: Cobamidas/metabolismo
Etanolamina Amônia-Liase/química
Etanolamina Amônia-Liase/metabolismo
Radicais Livres/metabolismo
Eletricidade Estática
[Mh] Termos MeSH secundário: Radicais Livres/química
Modelos Moleculares
Termodinâmica
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cobamides); 0 (Free Radicals); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase); F0R1QK73KB (cobamamide)
[Em] Mês de entrada:1405
[Cu] Atualização por classe:170922
[Lr] Data última revisão:
170922
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130821
[St] Status:MEDLINE
[do] DOI:10.1002/cbic.201300420


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[PMID]:23876477
[Au] Autor:Foti M; Médici R; Ruijssenaars HJ
[Ad] Endereço:B-Basic, Julianalaan 67, 2628 BC Delft, The Netherlands.
[Ti] Título:Biological production of monoethanolamine by engineered Pseudomonas putida S12.
[So] Source:J Biotechnol;167(3):344-9, 2013 Sep 10.
[Is] ISSN:1873-4863
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:Pseudomonas putida S12 was engineered for the production of monoethanolamine (MEA) from glucose via the decarboxylation of the central metabolite L-serine, which is catalyzed by the enzyme L-serine decarboxylase (SDC). The host was first evaluated for its tolerance towards MEA as well as its endogenous ability to degrade this alkanolamine. Growth inhibition was observed at MEA concentrations above 100 mM, but growth was never completely arrested even at 750 mM of MEA. P. putida S12 was able to catabolize MEA in the absence of ammonia, but deletion of the eutBC genes that encode ethanolamine ammonia-lyase (EAL) enzyme sufficed to eliminate this capacity. For the biological production of MEA, the sdc genes from Arabidopsis thaliana (full-length and a truncated version) and Volvox carteri were expressed in P. putida S12. From 20 mM of glucose, negligible amounts of MEA were produced by P. putida S12 ΔeutBC expressing the sdc genes from A. thaliana and V. carteri. However, 0.07 mmol of MEA was obtained per g of cell dry weight of P. putida S12 ΔeutBC expressing the truncated variant of the A. thaliana SDC. When the medium was supplemented with L-serine (30 mM), MEA production increased to 1.25 mmol MEA g⁻¹ CDW, demonstrating that L-serine availability was limiting MEA production.
[Mh] Termos MeSH primário: Etanolamina/metabolismo
Engenharia Genética/métodos
Pseudomonas putida/genética
[Mh] Termos MeSH secundário: Arabidopsis/genética
Proteínas de Bactérias/genética
Carboxiliases/genética
Carboxiliases/metabolismo
Etanolamina Amônia-Liase/genética
Deleção de Genes
Proteínas de Plantas/genética
Proteínas de Plantas/metabolismo
Pseudomonas putida/metabolismo
Volvox/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (Plant Proteins); 5KV86114PT (Ethanolamine); EC 4.1.1.- (Carboxy-Lyases); EC 4.1.1.- (L-serine decarboxylase); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase)
[Em] Mês de entrada:1403
[Cu] Atualização por classe:130902
[Lr] Data última revisão:
130902
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130724
[St] Status:MEDLINE


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[PMID]:23807763
[Au] Autor:Robertson WD; Bovell AM; Warncke K
[Ad] Endereço:Department of Physics, N201 Mathematics and Science Center, Emory University, 400 Dowman Drive, Atlanta, GA 30322-2430, USA.
[Ti] Título:Cobinamide production of hydrogen in a homogeneous aqueous photochemical system, and assembly and photoreduction in a (ßα)8 protein.
[So] Source:J Biol Inorg Chem;18(6):701-13, 2013 Aug.
[Is] ISSN:1432-1327
[Cp] País de publicação:Germany
[La] Idioma:eng
[Ab] Resumo:Components of a protein-integrated, earth-abundant metal macrocycle catalyst, with the purpose of H2 production from aqueous protons under green conditions, are characterized. The cobalt-corrin complex, cobinamide, is demonstrated to produce H2 (4.4 ± 1.8 × 10(-3) turnover number per hour) in a homogeneous, photosensitizer/sacrificial electron donor system in pure water at neutral pH. Turnover is proposed to be limited by the relatively low population of the gateway cobalt(III) hydride species. A heterolytic mechanism for H2 production from the cobalt(II) hydride is proposed. Two essential requirements for assembly of a functional protein-catalyst complex are demonstrated for interaction of cobinamide with the (ßα)8 TIM barrel protein, EutB, from the adenosylcobalamin-dependent ethanolamine ammonia lyase from Salmonella typhimurium: (1) high-affinity equilibrium binding of the cobinamide (dissociation constant 2.1 × 10(-7) M) and (2) in situ photoreduction of the cobinamide-protein complex to the Co(I) state. Molecular modeling of the cobinamide-EutB interaction shows that these features arise from specific hydrogen-bond and apolar interactions of the protein with the alkylamide substituents and the ring of the corrin, and accessibility of the binding site to the solution. The results establish cobinamide-EutB as a platform for design and engineering of a robust H2 production metallocatalyst that operates under green conditions and uses the advantages of the protein as a tunable medium and material support.
[Mh] Termos MeSH primário: Cobamidas/metabolismo
Etanolamina Amônia-Liase/química
Etanolamina Amônia-Liase/metabolismo
Hidrogênio/metabolismo
Processos Fotoquímicos
[Mh] Termos MeSH secundário: Sítios de Ligação
Cobalto/química
Cobalto/metabolismo
Cobamidas/química
Hidrogênio/química
Modelos Moleculares
Estrutura Molecular
Compostos Organometálicos/química
Compostos Organometálicos/metabolismo
Oxirredução
Salmonella typhimurium/metabolismo
Água/química
Água/metabolismo
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Cobamides); 0 (Organometallic Compounds); 059QF0KO0R (Water); 13497-85-3 (cobinamide); 3G0H8C9362 (Cobalt); 7YNJ3PO35Z (Hydrogen); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase)
[Em] Mês de entrada:1409
[Cu] Atualização por classe:170220
[Lr] Data última revisão:
170220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130629
[St] Status:MEDLINE
[do] DOI:10.1007/s00775-013-1015-3


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[PMID]:23374068
[Au] Autor:Bovell AM; Warncke K
[Ad] Endereço:Department of Physics, Emory University, Atlanta, Georgia 30322, United States.
[Ti] Título:The structural model of Salmonella typhimurium ethanolamine ammonia-lyase directs a rational approach to the assembly of the functional [(EutB-EutC)2]3 oligomer from isolated subunits.
[So] Source:Biochemistry;52(8):1419-28, 2013 Feb 26.
[Is] ISSN:1520-4995
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Ethanolamine ammonia-lyase (EAL) is a 5'-deoxyadenosylcobalamin-dependent bacterial enzyme that catalyzes the deamination of the short-chain vicinal amino alcohols, aminoethanol and (S)- and (R)-2-aminopropanol. The coding sequence for EAL is located within the 17-gene eut operon, which encodes the broad spectrum of proteins that comprise the ethanolamine utilization (eut) metabolosome suborganelle structure. A high-resolution structure of the ∼500 kDa EAL [(EutB-EutC)2]3 oligomer from Escherichia coli has been determined by X-ray crystallography, but high-resolution spectroscopic determinations of reactant intermediate-state structures and detailed kinetic and thermodynamic studies of EAL have been conducted for the Salmonella typhimurium enzyme. Therefore, a statistically robust homology model for the S. typhimurium EAL is constructed from the E. coli structure. The model structure is used to describe the hierarchy of EutB and EutC subunit interactions that construct the native EAL oligomer and, specifically, to address the long-standing challenge of reconstitution of the functional oligomer from isolated, purified subunits. Model prediction that the (EutB2)3 oligomer assembly will occur from isolated EutB, and that this hexameric structure will template the formation of the complete, native [(EutB-EutC)2]3 oligomer, is verified by biochemical methods. Prediction that cysteine residues on the exposed subunit-subunit contact surfaces of isolated EutB and EutC will interfere with assembly by cystine formation is verified by activating effects of disulfide reducing agents. Angstrom-scale congruence of the reconstituted and native EAL in the active site region is shown by electron paramagnetic resonance spectroscopy. Overall, the hierarchy of subunit interactions and microscopic features of the contact surfaces, which are revealed by the homology model, guide and provide a rationale for a refined genetic and biochemical approach to reconstitution of the functional [(EutB-EutC)2]3 EAL oligomer. The results establish a platform for further advances in understanding the molecular mechanism of EAL catalysis and for insights into therapy-targeted manipulation of the bacterial eut metabolosome.
[Mh] Termos MeSH primário: Etanolamina Amônia-Liase/química
Etanolamina Amônia-Liase/metabolismo
Salmonella typhimurium/enzimologia
[Mh] Termos MeSH secundário: Cobamidas/metabolismo
Espectroscopia de Ressonância de Spin Eletrônica
Escherichia coli/química
Escherichia coli/enzimologia
Etanolamina Amônia-Liase/genética
Modelos Moleculares
Mutagênese Sítio-Dirigida
Plasmídeos/genética
Conformação Proteica
Multimerização Proteica
Subunidades Proteicas/química
Subunidades Proteicas/genética
Subunidades Proteicas/metabolismo
Salmonella typhimurium/química
Salmonella typhimurium/genética
Homologia Estrutural de Proteína
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL; RESEARCH SUPPORT, U.S. GOV'T, NON-P.H.S.
[Nm] Nome de substância:
0 (Cobamides); 0 (Protein Subunits); EC 4.3.1.7 (Ethanolamine Ammonia-Lyase); F0R1QK73KB (cobamamide)
[Em] Mês de entrada:1304
[Cu] Atualização por classe:170220
[Lr] Data última revisão:
170220
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:130205
[St] Status:MEDLINE
[do] DOI:10.1021/bi301651n



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