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

Margaret McAuley, Noel Mesa-Torres, Aisling McFall, Sarah Morris, Meilan Huang, Angel L. Pey, David J. Timson

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5 Citations (Scopus)
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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 galactosemia. The return-to-consensus approach can be used to identify residues which 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 increased the catalytic turnover of the enzyme (M60V, D268E, A334S and G373S), 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 may also be useful if the protein were to be used in ERT.
Original languageEnglish
Pages (from-to)1088-1095
JournalChemBioChem
Volume19
Issue number10
Early online date05 Mar 2018
DOIs
Publication statusPublished - 18 May 2018

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