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Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.
Taillefer, M; Rydzak, T; Levin, D B; Oresnik, I J; Sparling, R.
Afiliação
  • Taillefer M; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
  • Rydzak T; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
  • Levin DB; Department of Biosystems Engineering, University of Manitoba, Winnipeg, Manitoba, Canada.
  • Oresnik IJ; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
  • Sparling R; Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada Richard.Sparling@umanitoba.ca.
Appl Environ Microbiol ; 81(7): 2423-32, 2015 Apr.
Article em En | MEDLINE | ID: mdl-25616802
Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Clostridium thermocellum / Redes e Vias Metabólicas / Malato Desidrogenase / Malatos / NADP Trans-Hidrogenases Idioma: En Revista: Appl Environ Microbiol Ano de publicação: 2015 Tipo de documento: Article País de afiliação: Canadá País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Clostridium thermocellum / Redes e Vias Metabólicas / Malato Desidrogenase / Malatos / NADP Trans-Hidrogenases Idioma: En Revista: Appl Environ Microbiol Ano de publicação: 2015 Tipo de documento: Article País de afiliação: Canadá País de publicação: Estados Unidos