Activity improvement of UDP-galactose-4-epimerase for tagatose substrates by 3D structure-based combinatorial mutagenesis

Uridine diphosphogalactose-4-epimerase (UDP-galactose-4-epimerase, GalE, EC 5.1.3.2) mediates 4-epimerization of free monosaccharides in a low activity as well as original UDP-galactose substrate in a high activity (Appl Biochem Biotechnol 163:444–451). To improve GalE for monosaccharide 4-epimerization, random mutations were introduced and the effects of single mutations on the conversion of tagatose were evaluated. Among 3060 random mutated GalE variants, two clones showing the improved conversion activity of tagatose 4-epimerization were selected and their mutation points were sequenced. To investigate the effect of each mutations, the 5 mutation points (D58E, N100S, P193S, I196N, and T317S) found in the two selected clones were further introduced into the wild-type GalE and the variant enzymes were purified for kinetic study. The enzyme carrying D58E single mutation (GalE D58E), which is located near the cofactor binding pocket in a 3D structure docking model, showed the kinetic parameters of KM = 388 mM and kcat = 1.77 min−1 for tagatose substrate and it was 3.3-fold greater kcat/KM than the wild-type enzyme. When the N179S mutation, which is located near the substrate-binding pocket according to our previous study, was further combined with GalE D58E, the kinetic parameters of the double mutant enzyme was found to have 3.7-fold higher kcat/KM than the wild-type. The effect of the mutations on the activity improvement based on the 3D structure docking model and further biotechnological potential is discussed.

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► Monosaccharide 4-epimerization reaction was improved by UDP-galactose-4-epimerase (GalE).
► Mutations near cofactor binding site (Glu 58 and Ser 100) of the enzyme contributed the increase of turnover.
► Combinatorial mutation (Ser 179) near substrate binding site of the enzyme contributed the increase of substrate affinity.