RESUMO
The BaM6PI gene encoding a mannose-6-phosphate isomerase (M6PI, EC 5.3.1.8) was cloned from Bacillus amyloliquefaciens DSM7 and overexpressed in Escherichia coli. The enzyme activity of BaM6PI was optimal at pH and temperature of 7.5 and 70°C, respectively, with a kcat/Km of 13,900 s-1 mM-1 for mannose-6-phosphate (M6P). The purified BaM6PI demonstrated the highest catalytic efficiency of all characterized M6PIs. Although M6PIs have been characterized from several other sources, BaM6PI is distinguished from other M6PIs by its wide pH range and high catalytic efficiency for M6P. The binding orientation of the substrate M6P in the active site of BaM6PI shed light on the molecular basis of its unusually high activity. BaM6PI showed 97% substrate conversion from M6P to fructose-6-phosphate demonstrating the potential for using BaM6PI in industrial applications.
Assuntos
Bacillus/enzimologia , Frutosefosfatos/biossíntese , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Sequência de Aminoácidos , Bacillus/genética , Clonagem Molecular , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Manose-6-Fosfato Isomerase/química , Metais/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peso Molecular , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity.
Assuntos
Proteínas de Bactérias/metabolismo , Enterobacter aerogenes/enzimologia , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biotecnologia , Domínio Catalítico , Enterobacter aerogenes/genética , Genes Bacterianos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribitol/metabolismo , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Especificidade por Substrato , Desidrogenase do Álcool de Açúcar/química , Desidrogenase do Álcool de Açúcar/genéticaRESUMO
Soluble methane monooxygenase (sMMO) is a bacterial multicomponent enzyme that oxidizes a diverse range of substrates, including aromatic hydrocarbons. We have investigated enzyme-substrate interactions that govern oxidation regioselectivity at various sites of aromatic compounds using substrate docking and molecular dynamics (MD) simulations. Here, we studied the hydroxylation of toluene and ethyl benzene by two forms of Methylosinus trichosporium OB3b (sMMO), that is, wild-type (WT) and two active site mutants (L110Y/G). The two substrates, toluene and ethyl benzene, were docked into the active site of the WT and the L110Y/G mutant models of M. trichosporium OB3b sMMO using the available X-ray structure (PDB id 1 MHZ). The trends observed in the formation of the experimental product were highly correlated with the results obtained from the relatively short MD simulation. These results show that our approach could be an attractive computational tool to rationalize the prediction of product ratios and specificities.
Assuntos
Hidrocarbonetos Aromáticos/metabolismo , Simulação de Dinâmica Molecular , Oxigenases/metabolismo , Hidrocarbonetos Aromáticos/química , Hidroxilação , Methylosinus trichosporium/química , Modelos Moleculares , Estrutura Molecular , Oxigenases/química , Solubilidade , EstereoisomerismoRESUMO
Acetate and lactate in growth media are detrimental to the production of Thermus maltogenic amylase (ThMA), a heterologous protein, as well as to the growth of recombinant Escherichia coli. Only 50 mM of acetate or 10 mM of lactate reduced 90% of specific ThMA activity. In this study, mutant E. coli strains blocked in the ackA-pta or ackA-pta and ldh pathways were created, characterized, and assessed for their culture performace in 300 L-scale fermentation. The ackApta and ldh double-mutant strain formed significantly less lactate and acetate, and produced a concomitant increase in the excretion of pyruvate (17.8 mM) under anaerobic conditions. The ackA-pta mutant strain accumulated significant acetate but had an approximately 2-fold increase in the formation of lactate. The ackA-pta and ldh double-mutant strain had superior overall performance in large-scale culture under suboptimal conditions, giving 67% higher cell density and 66% higher ThMA activity compared with those of the control strain. The doublemutant strain also achieved a 179% improvement in volumetric ThMA production.