Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications

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.

Modulation of the mobility of a key region in human galactokinase: Impacts on catalysis and stability

“…Molecular dynamics (MD) simulations were carried out essentially as previously described [6,8] using Amber 10 and the Amber Parm 99 forcefield [38]. The crystal structure of human galactokinase (PDB: 1WUU; chain A) was used as the basis for all simulations [39].…”

“…Human galactokinase was expressed in, and purified from, E. coli HMS174(DE3) as previously described [6,8,9,23,52]. Briefly, cultures (1 l) of recombinant bacteria were grown, lysed by sonication and galactokinase purified from the resulting extract by cobalt affinity chromatography (His-Select, Sigma, UK).…”

“…Galactokinase activity was assayed by coupling the production of ADP to the reactions catalysed by pyruvate kinase (EC 2.7.1.4) and lactate dehydrogenase (EC 1.1.1.27). This enables the reaction to be followed via the consumption of NADH and the consequent reduction in absorbance at 340 nm [6,8,54]. Reactions were carried out in a total volume of 160 µl in 96 well plates at 37 °C.…”

“…We have previously shown that several regions which are not part of the active site are critical for controlling specificity and catalysis in human galactokinase (EC 2.7.1.6) [6][7][8][9][10]. This enzyme is a member of the GHMP kinase family and catalyses the first committed step of the Leloir pathway, the ATP-dependent phosphorylation of α-D-galactose [11][12][13].…”

“…This enzyme is a member of the GHMP kinase family and catalyses the first committed step of the Leloir pathway, the ATP-dependent phosphorylation of α-D-galactose [11][12][13]. The human enzyme is relatively highly selective -only α-D-galactose, α-D-galactosamine, 2-deoxy-α-D-galactose and α-D-talose are known to be substrates [8,9,14]. One region, located around Try-379, influences the flexibility of residues which form the active site.…”