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

preprint
revised on 29.09.2020 and posted on 30.09.2020 by Henry P Wood, Nicola J Baxter, Clare R Trevitt, F Aaron Cruz-Navarrete, Andrea M Hounslow, Jonathan P Waltho

Manipulation of enzyme behaviour represents a sustainable technology that can be harnessed to enhance the production of valuable metabolites and chemical precursors. b-glucose 1,6-bisphosphate (bG16BP) is a native reaction intermediate in the catalytic cycle of b-phosphoglucomutase (bPGM) that has been proposed as a treatment for human congenital disorder of glycosylation involving phosphomannomutase 2 (PMM2). Studies of both bPGM and PMM2 could benefit from a green and high-yielding method for bG16BP production. Three strategies have been reported previously for the synthesis of bG16BP; however, each of these methods either delivers low yields or uses chemicals and procedures with significant environmental impacts. Herein, through combined use of NMR spectroscopy, kinetic assays and site-directed mutagenesis, we report the efficient enzymatic synthesis of anomer-specific bG16BP using a variant of bPGM. Further purification, employing a simple environmentally considerate precipitation procedure requiring only a standard biochemical toolset, results in a product with high purity and yield. Moreover, 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.

Funding

BBSRC X/009906-20-26

BBSRC BB/M021637/1

BBSRC BB/S007965/1

BBSRC BB/P007066/1

CONACYT 472448

History

Email Address of Submitting Author

j.waltho@sheffield.ac.uk

Institution

University of Manchester

Country

United Kingdom

ORCID For Submitting Author

0000-0002-7402-5492

Declaration of Conflict of Interest

There are no conflicts to declare.

Version Notes

Version 2.0

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