Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
FEBS J ; 277(24): 5124-32, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21078123

RESUMO

The crystal structure of a L-threonine dehydrogenase (L-ThrDH; EC 1.1.1.103) from the psychrophilic bacterium Flavobacterium frigidimaris KUC-1, which shows no sequence similarity to conventional L-ThrDHs, was determined in the presence of NAD and a substrate analog, glycerol. The asymmetric unit consisted of two subunits related by a two-fold rotation axis. Each monomer consisted of a Rossmann-fold domain and a carboxyl-terminal catalytic domain. The overall fold of F. frigidimaris L-ThrDH showed significant similarity to that of UDP-galactose 4-epimerase (GalE); however, structural comparison of the enzyme with E. coli and human GalEs showed clear topological differences in three loops (loop 1, loop 2 and the NAD-binding loop) around the substrate and NAD binding sites. In F. frigidimaris L-ThrDH, loops 1 and 2 insert toward the active site cavity, creating a barrier preventing the binding of UDP-glucose. Alternatively, loop 1 contributes to a unique substrate binding pocket in the F. frigidimaris enzyme. The NAD binding loop, which tightly holds the adenine ribose moiety of NAD in the Escherichia coli and human GalEs, is absent in F. frigidimaris L-ThrDH. Consequently, the cofactor binds to F. frigidimaris L-ThrDH in a reversible manner, unlike its binding to GalE. The substrate binding model suggests that the reaction proceeds through abstraction of the ß-hydroxyl hydrogen of L-threonine via either a proton shuttle mechanism driven by Tyr143 and facilitated by Ser118 or direct proton transfer driven by Tyr143. The present structure provides a clear bench mark for distinguishing GalE-like L-ThrDHs from GalEs.


Assuntos
Oxirredutases do Álcool/química , UDPglucose 4-Epimerase/química , Oxirredutases do Álcool/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/enzimologia , Flavobacterium/enzimologia , Humanos , Modelos Moleculares , Conformação Proteica , Treonina/metabolismo , UDPglucose 4-Epimerase/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-18007057

RESUMO

Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 A. It contains one tetrameric molecule in the asymmetric unit.


Assuntos
Flavobacterium/enzimologia , Malato Desidrogenase/química , Regiões Antárticas , Temperatura Baixa , Cristalização , Cristalografia por Raios X , Estrutura Quaternária de Proteína
3.
Extremophiles ; 11(2): 257-67, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17072683

RESUMO

An NAD(+)-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD(+) as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD(+). F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85 degrees C and the most active at 70 degrees C. The half-life time and k (cat) value at 60 degrees C were calculated to be 50 min and 27,400 min(-1), respectively. The enzyme also shows high catalytic efficiency at low temperatures (0-20 degrees C) (k(cat)/K(m) at 10 degrees C; 12,600 mM(-1)min(-1)) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.


Assuntos
Álcool Desidrogenase/química , Álcool Desidrogenase/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Flavobacterium/enzimologia , Microbiologia da Água , Álcool Desidrogenase/genética , Regiões Antárticas , Proteínas de Bactérias/genética , Sequência de Bases , Catálise , Temperatura Baixa , Estabilidade Enzimática , Temperatura Alta , Dados de Sequência Molecular , Água do Mar/microbiologia
4.
J Bacteriol ; 185(15): 4483-9, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12867457

RESUMO

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD(+)-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degrees C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K(m) for L-threonine was lowest at 20 degrees C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.


Assuntos
Oxirredutases do Álcool , Cytophaga/enzimologia , Água do Mar/microbiologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Animais , Regiões Antárticas , Clonagem Molecular , Temperatura Baixa , Cytophaga/isolamento & purificação , Estabilidade Enzimática , Temperatura Alta , Humanos , Cinética , Dados de Sequência Molecular , NAD/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , UDPglucose 4-Epimerase/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...