RESUMO
Indole-3-glycerol phosphate synthase (IGPS) catalyzes the irreversible ring closure of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), through decarboxylation and dehydration steps, releasing indole-3-glycerol phosphate (IGP), the fourth step in the biosynthesis of tryptophan. This pathway is essential for Mycobacterium tuberculosis virulence. Here we describe the cloning, expression, purification, and kinetic characterization of IGPS from M. tuberculosis. To perform kinetic studies, CdRP was chemically synthesized, purified, and spectroscopically and spectrometrically characterized. CdRP fluorescence was pH-dependent, probably owing to excited-state intramolecular proton transfer. The activation energy was calculated, and solvent isotope effects and proton inventory studies were performed. pH-rate profiles were carried out to probe for acid/base catalysis, showing that a deprotonated residue is necessary for CdRP binding and conversion to IGP. A model to describe a steady-state kinetic sequence for MtIGPS-catalized chemical reaction is proposed.
Assuntos
Indol-3-Glicerolfosfato Sintase/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Bases , Fenômenos Biofísicos , Clonagem Molecular , Primers do DNA/genética , DNA Bacteriano/genética , Genes Bacterianos , Concentração de Íons de Hidrogênio , Indol-3-Glicerolfosfato Sintase/genética , Indol-3-Glicerolfosfato Sintase/isolamento & purificação , Cinética , Espectroscopia de Ressonância Magnética , Modelos Químicos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Ribulosefosfatos/síntese química , Ribulosefosfatos/metabolismo , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Termodinâmica , VirulênciaRESUMO
A novel scheme employing enzymatic catalysts is described enabling conversion of D-ribulose-1,5-bisphosphate (RuBP) from 3-phospho-D-glycerate (3-PGA) without loss of carbon. Bioreactors harboring immobilized enzymes namely, phosphoglycerate kinase (PGK), glycerate phosphate dehydrogenase, triose phosphate isomerase (TIM), aldolase, transketolase (TKL), phosphatase (PTASE/FP), epimerase (EMR) and phosphoribulokinase (PRK), in accordance with this novel scheme were employed. These reactors were designed and constructed based on simulations carried out to study their performance under various operational conditions and allowed production of about 56 +/- 3% RuBP from 3-PGA. This method of synthesis of RuBP from 3-PGA employing immobilized enzyme bioreactors may be used for continuous regeneration of RuBP in biocatalytic carbon dioxide fixation processes from emissions where RuBP acts as acceptor of carbon dioxide to produce 3-PGA, rendering the fixation process continuous.
Assuntos
Reatores Biológicos , Enzimas Imobilizadas/química , Ácidos Glicéricos/química , Modelos Químicos , Complexos Multienzimáticos/química , Ribulosefosfatos/síntese química , Catálise , Simulação por Computador , Desenho Assistido por Computador , Desenho de Equipamento/métodos , CinéticaRESUMO
The production of D-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to D-ribulose was catalysed by D-ribulokinase isolated from Klebsiella pneumoniae. For production of D-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for D-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of D-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of D-ribulose-5-phosphate l -1 per day were reached.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Klebsiella pneumoniae/enzimologia , Pentosefosfatos/síntese química , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/isolamento & purificação , Ribulosefosfatos/síntese química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Fosfotransferases/metabolismo , Piruvato Quinase/metabolismoRESUMO
A procedure is described to prepare uniformly labelled D-[14C]ribulose 1,5-bisphosphate enzymically from uniformly labelled D-[14C]glucose through the coupled reactions catalysed by hexokinase (EC 2.7.1.1), glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and 5-phosphoribulokinase (EC 2.7.1.19). All reagents utilized in the method are commercially available. The procedure is a reliable preparative-scale method for synthesizing the dibarium salt of D-[14C]ribulose 1,5-biphosphate with a specific radioactivity up to 7 mCi/mmol and a purity near 90%. The final product was free of other 14C-labelled sugars, sugar phosphate esters, Pi and nucleotides.
