Wood, H.P., Baxter, N.J., Cruz-Navarrete, F.A. et al. (3 more authors) (2021) Enzymatic production of β-glucose 1,6-bisphosphate through manipulation of catalytic magnesium coordination. Green Chemistry, 23 (2). pp. 752-762. ISSN 1463-9262
Abstract
Manipulation of enzyme behaviour represents a sustainable technology that can be harnessed to enhance the production of valuable metabolites and chemical precursors. β-Glucose 1,6-bisphosphate (βG16BP) is a native reaction intermediate in the catalytic cycle of β-phosphoglucomutase (βPGM) that has been proposed as a treatment for human congenital disorder of glycosylation involving phosphomannomutase 2. Strategies to date for the synthesis of βG16BP suffer from low yields or use chemicals and procedures with significant environmental impacts. Herein, we report the efficient enzymatic synthesis of anomer-specific βG16BP using the D170N variant of βPGM (βPGMD170N), 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 βPGMD170N towards βG16BP contributes to the pronounced retardation of the second step in the two-step catalytic cycle, which causes a marked accumulation of βG16BP, especially at elevated MgCl2 concentrations. Purification, employing a simple environmentally considerate precipitation procedure requiring only a standard biochemical toolset, results in a βG16BP 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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Royal Society of Chemistry 2021. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. (http://creativecommons.org/licenses/by/3.0/) |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 09 Mar 2021 12:11 |
Last Modified: | 09 Mar 2021 12:11 |
Status: | Published |
Publisher: | Royal Society of Chemistry (RSC) |
Refereed: | Yes |
Identification Number: | 10.1039/d0gc03290e |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:171281 |