Enzymatic Production of Β-Glucose 1,6-Bisphosphate Through Manipulation of Catalytic Magnesium Coordination

Manipulation of enzyme behaviourrepresents a sustainable technology that can be harnessed to enhance theproduction of valuable metabolites and chemical precursors. b-glucose 1,6-bisphosphate (bG16BP) is a native reactionintermediate in the catalytic cycle of b-phosphoglucomutase(bPGM) that has been proposed as a treatmentfor human congenital disorder of glycosylation involving phosphomannomutase 2(PMM2). Studies of both bPGMand PMM2 could benefit from a green and high-yielding method for bG16BP production. Three strategieshave been reported previously for the synthesis of bG16BP; however, each of thesemethods either delivers low yields or uses chemicals and procedures with significantenvironmental impacts. Herein, through combined use of NMR spectroscopy, kinetic assays and site-directed mutagenesis, we report the efficient enzymatic synthesis of anomer-specificbG16BP using a variant of bPGM. Further purification,employing a simple environmentally considerate precipitation procedurerequiring only a standard biochemical toolset, results in a product with high purityand yield. Moreover, this synthesis strategy illustrates how manipulation ofthe catalytic magnesium coordination of an enzyme can be utilised to generate largequantities of a valuable metabolite.