Genetic Evidence Identifying the True Gluconeogenic Fructose-1,6-Bisphosphatase in Thermococcus kodakaraensis and Other Hyperthermophiles

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Journal of Bacteriology (Impact Factor: 2.81). 10/2004; 186(17):5799-807. DOI: 10.1128/JB.186.17.5799-5807.2004
Source: PubMed


Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected
in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified
in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the “missing” FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog
in T. kodakaraensis, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore
constructed Δfbp and Δimp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Δfbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted
in the complete abolishment of intracellular FBPase activity. Evidently, fbpTk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast,
the endogenous impTk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts
indicated that impTk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription.
Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the FbpTk ortholog, not the IMPase/FBPase ortholog.

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Available from: Naeem Rashid
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    • "The genome harbors pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) and proton-translocating pyrophosphatase, which have been identified in methanotrophic and thiotrophic Gammaproteobacteria [35], [55], [56]. However, typical bacterial fructose 1,6-bisphosphatases (class 1 and 2) and their possible alternatives, such as archaeal ADP-dependent phosphofructokinase [57] and recently identified fructose 1,6 bisphosphate aldolase/phosphatase [58], were not identified. We checked the enzymatic activities of bacterial 6-phosphofructokinases and fructose 1,6-bisphosphatases in the cell-free extract prepared from cells grown chemolithoautotrophically. Consumption and production activities of fructose 6-phoshate by PPi-PFK were determined to be 0.66 and 0.15 µmol/min/mg protein, respectively, in the cell-free extract at 37°C. "
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    • "New avenues, based on advances in T. kodakarensis genetics, permit direct characterization of innumerable archaeal enzymes and their chemistries (Atomi et al., 2001, 2004a; Shiraki et al., 2003; Fukuda et al., 2004, 2008; Rashid et al., 2004; Sato et al., 2004, 2007; Imanaka et al., 2006; Murakami et al., 2006; Orita et al., 2006; Kanai et al., 2007, 2010, 2011; Danno et al., 2008; Fujiwara et al., 2008; Louvel et al., 2009; Yokooji et al., 2009; Borges et al., 2010; Kobori et al., 2010; Morimoto et al., 2010; Matsubara et al., 2011), provide industrially relevant alternative biofuel platforms(Kanai et al., 2005, 2011; Chou et al., 2008; Kim et al., 2010; Atomi et al., 2011; Santangelo et al., 2011; Bae et al., 2012; Davidova et al., 2012), unlock the largely untapped reservoir of archaeal encoded natural products (Atomi, 2005; Kim and Peeples, 2006; Littlechild, 2011; Matsumi et al., 2011; Sato and Atomi, 2011), and offer the opportunity to dissect eukaryotic-like information processing systems composed of minimal components (Yamamoto et al., 2003; Santangelo and Reeve, 2006, 2010a; Kanai et al., 2007; Santangelo et al., 2007, 2008a, 2009; Hirata et al., 2008; Dev et al., 2009; Yamaji et al., 2009; Fujikane et al., 2010; Li et al., 2010, 2011; Ishino et al., 2011; Nunoura et al., 2011; Pan et al., 2011; Santangelo and Artsimovitch, 2011). "
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