Characterization of key catabolic enzymes in the central catalysis of Ruminiclostridium thermocellum reveals alternative cofactor utilisation and enzyme regulation

Ruminiclostridium thermocellum produces ethanol as a major end product from direct fermentation of cellulosic biomass.  Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing.  A deeper understanding of the regulatory mechanism controlling the metabolic flux through glycolysis and how glycolytic flux regulation modulates end product yields may reveal attractive targets for metabolic engineering.  High-energy metabolites, such as ATP, GTP, and pyrophosphate, can directly interact with key catabolic enzymes modulating their activities as cofactors or allosteric activators/inhibitors.  We describe the biochemical characterization of His-tagged purified enzymes predicted to be key contributors of catabolic flux in the central catalysis in R. thermocellum ATCC27405 such as glucokinase (Cthe_2938), phosphofructokinase (Cthe_0347), and phosphoglycerate kinase (Cthe_0138).  The glucokinase was found to be a bacterial type glucokinase specific for glucose.  The glucokinase exhibited strong preference for the utilisation of GTP over ATP as phosphate donors with no observable activity found using pyrophosphate. However, pyrophosphate was found to increase glucokinase activity at 50^oC.  Conversely, increasing concentrations of GTP and/or ATP resulted in strong inhibition of glucokinase activity passed a peak of activity found around 1mM.  This suggests a putative regulatory mechanism utilized in the maintenance of relatively low intracellular concentrations of GTP and/or ATP favouring the build-up of alternative energy storage molecules such as pyrophosphate.  Therefore, careful understanding of the fluctuations of high energy metabolites along with the regulatory mechanisms modulating flux through glycolysis will help elucidate the optimal internal conditions required for cellular growth and end products synthesis.