Novel Activity of UDP-Galactose-4-Epimerase for Free Monosaccharide and Activity Improvement by Active Site-Saturation Mutagenesis |
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Authors: | Hye-Jung Kim Sueng Yeun Kang Jong Jin Park Pil Kim |
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Institution: | (1) Department of Biotechnology, Catholic University of Korea, Bucheon, Gyeonggi, 420-743, South Korea;(2) Food & Bio Research Center, Samyang Genex Co., Yuseong, Daejon, 305-717, South Korea; |
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Abstract: | Uridine diphosphogalactose-4-epimerase (UDP-galactose-4-epimerase, GalE, EC 5.1.3.2) mediates the 4-epimerization of nucleic
acid-activated galactose into UDP-glucose. To date, no enzyme is known to mediate 4-epimerization of free monosaccharide substrates.
To determine the potential activity of GalE for free monosaccharide, Escherichia coli GalE was expressed and purified using Ni-affinity chromatography, and its ability to mediate 4-epimerization of a variety
of free keto- and aldohexoses was assessed. Purified GalE was found to possess 4-epimerization activity for free galactose,
glucose, fructose, tagatose, psicose, and sorbose at 0.47, 0.31, 2.82, 9.67, 15.44, and 2.08 nmol/mg protein per minute, respectively.
No 4-epimerization activity was found for allose, gulose, altrose, idose, mannose, and talose. The kinetic parameters of 4-epimerization
reactions were K
m = 26.4 mM and k
cat = 0.0155 min−1 for d-galactose and K
m = 237 mM and k
cat = 0.327 min−1 for d-tagatose. The 4-epimerization of tagatose, a reaction of commercial interest, was enhanced twofold (19.79 nmol/mg protein
per minute) when asparagine was exchanged with serine at position 179. The novel activity of GalE for free monosaccharide
may be beneficial for the production of rare sugars using cheap natural resources. Potential strategies for developing enhanced
GalE with increased 4-epimerization activity are discussed in the context of the above findings and an analysis of a 3D structural
model. |
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