Enzymatic Production Of Beta-Glucose 1,6-Bisphosphate Through Manipulation Of Catalytic Magnesium Coordination

Manipulation of enzyme behaviour represents a sustainable technology that can be harnessed to enhance the production of valuable metabolites and chemical precursors. beta-Glucose 1,6-bisphosphate (beta G16BP) is a native reaction intermediate in the catalytic cycle of beta-phosphoglucomutase (beta PGM) that has been proposed as a treatment for human congenital disorder of glycosylation involving phosphomannomutase 2. Strategies to date for the synthesis of beta G16BP suffer from low yields or use chemicals and procedures with significant environmental impacts. Herein, we report the efficient enzymatic synthesis of anomer-specific beta G16BP using the D170N variant of beta PGM (beta PGM(D170N)), where the aspartate to asparagine substitution at residue 170 perturbs the coordination of a catalytic magnesium ion. Through combined use of NMR spectroscopy and kinetic assays, it is shown that the weakened affinity and reactivity of beta PGM(D170N) towards beta G16BP contributes to the pronounced retardation of the second step in the two-step catalytic cycle, which causes a marked accumulation of beta G16BP, especially at elevated MgCl2 concentrations. Purification, employing a simple environmentally considerate precipitation procedure requiring only a standard biochemical toolset, results in a beta G16BP product with high purity and yield. Overall, this synthesis strategy illustrates how manipulation of the catalytic magnesium coordination of an enzyme can be utilised to generate large quantities of a valuable metabolite.