The bioconversion of inositol from glucose was
The bioconversion of inositol from glucose was anticipated nearly a century ago and later confirmed by isotope tracing methods [, ]. This biosynthesis of inositol involves three sequentially acting enzymes: Firstly, ATP-dependent glucokinase converts glucose to glucose-6-phosphate (G-6P) [, ]. Then, IPS catalyzes the conversion of G-6P to inositol-3-phosphate (I-3P) [, , , , ]. Finally, IMP dephosphorylates I-3P to generate inositol [, , , ]. The enzyme responsible for the rate-limiting step of inositol synthesis is IPS, which plays a crucial role in inositol homeostasis and exists in a wide range of plants, animals, and fungi [, ]. It has been reported that IPS converts G-6P to Ins-3P in a complex, four-step catalytic reaction [, ].
Polyphosphate glucokinase (PPGK), which uses polyphosphate [(Pi)n] as the phosphoryl donor, has shown great potential for G-6P generation without ATP supplementation [, , , ]. Therefore, we developed a novel pathway to produce inositol from glucose by a trienzymatic cascade involving PPGK, IPS and IMP (Fig. 1). Three highly active enzymes, AspPPGK from Arthrobacter sp. OY3WO11, TbIPS from Trypanosoma brucei TREU927, and EcIMP from Escherichia coli, were obtained via enzyme selection. The pathway showed 90% conversion of glucose to inositol and production of 45 mM inositol in the trienzymatic two-step cascade reaction.
Materials and methods
Results and discussion
Introduction Glucokinase (GK) plays a key role in the regulation of glucose homeostasis by catalyzing the first and rate limiting step in glycolysis (Meglasson and Matschinsky, 1984, Lenzen, 2014). Its expression is limited to glucose-sensitive tissues, notably pancreatic beta-cells, hepatocytes and neuroendocrine Lonidamine (Jetton et al., 1994). Different mechanisms regulate the expression of GK in these cells, related to cell-specific promoters within the GK encoding gene (GCK) (Bedoya et al., 1986, Iynedjian, 1993). In pancreatic beta-cells, GK expression is regulated by α-D-glucose, while in hepatocytes its expression is regulated by insulin. The enzyme is further activated by glucose binding, which in hepatocytes results in a stimulation of glucose uptake, glycolysis and glycogen synthesis, whereas in pancreatic beta-cells the result is glucose-stimulated insulin secretion (Matschinsky, 1990). Genetic studies on Mendelian disorders of glucose homeostasis have provided unequivocal evidence for the critical metabolic role of GK. Heterozygous inactivating GCK mutations result in a subtype of maturity-onset diabetes of the young (GCK-MODY or MODY2), while homozygous inactivating mutations produce permanent neonatal diabetes (Njolstad et al., 2001, Sagen et al., 2006). Conversely, rare heterozygous activating mutations cause familial hyperinsulinemic hypoglycemia (GCK-HH) (Glaser et al., 1998). A complex network of mechanisms has been reported for the posttranslational regulation of GK localization and function, and it is fundamentally different in pancreatic beta-cells and liver hepatocytes (Lenzen, 2014, Agius, 2008). In hepatocytes, GK is regulated by the glucokinase regulatory protein (GKRP), a competitive inhibitor of glucose binding to GK (Van Schaftingen, 1989). At low glucose concentrations, GKRP forms a complex with GK and shuttles the enzyme to the nucleus (Agius et al., 1996, de la Iglesia et al., 1999). When glucose levels rise, the GK-GKRP complex dissociates and GK returns to the cytosol to participate in glucose metabolism. Although GKRP is not expressed in pancreatic beta-cells, GK was found to translocate to the beta-cell nuclei under the influence of exogenous GKRP (Bosco et al., 2000). Moreover, in beta-cells, S-nitrosylation has been shown to regulate GK association with insulin secretory granules (Rizzo and Piston, 2003).