Improving the activity and stability of human galactokinase for therapeutic and biotechnological applications

McAuley, M., Mesa-Torres, N., McFall, A. , Morris, S., Huang, M., Pey, A. L. and Timson, D. J. (2018) Improving the activity and stability of human galactokinase for therapeutic and biotechnological applications. ChemBioChem, 19(10), pp. 1088-1095. (doi: 10.1002/cbic.201800025) (PMID:29505688)

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Abstract

Galactokinase catalyses the site- and stereospecific phosphorylation of α-d-galactose. As such it has attracted interest as a biocatalyst for the introduction of phosphate groups into monosaccharides. However, attempts to broaden the substrate range of human galactokinase have generally resulted in substantially reduced activity. The enzyme also has biotechnological potential in enzyme replacement therapy (ERT) for type II galactosaemia. The return-to-consensus approach can be used to identify residues that can be altered to increase protein stability and enzyme activity. This approach identified six residues of potential interest in human galactokinase. Some of the single consensus variants (M60V, D268E, A334S and G373S) increased the catalytic turnover of the enzyme, but none resulted in improved stability. When all six changes were introduced into the protein (M60V/M180V/D268E/A334S/R366Q/G373S), thermal stability was increased. Molecular dynamics simulations suggested that these changes altered the protein's conformation at key sites. The number of salt bridges and hydrogen bonds was also increased. Combining the six consensus variations with Y379W (a variant with greater substrate promiscuity) increased the stability of this variant and its turnover towards some substrates. Thus, the six consensus variants can be used to stabilise catalytically interesting variants of human galactokinase and might also be useful if the protein were to be used in ERT.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McFall, Dr Aisling
Authors: McAuley, M., Mesa-Torres, N., McFall, A., Morris, S., Huang, M., Pey, A. L., and Timson, D. J.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:ChemBioChem
Publisher:Wiley
ISSN:1439-4227
ISSN (Online):1439-7633
Published Online:05 March 2018

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