Novel activity of UDP-galactose-4-epimerase for free monosaccharide and activity improvement by active site-saturation mutagenesis.
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.
Kim
Hye-Jung
HJ
Department of Biotechnology, Catholic University of Korea, Bucheon, Gyeonggi, South Korea.
Kang
Sueng Yeun
SY
Park
Jong Jin
JJ
Kim
Pil
P
D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D005690:Galactose / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism; D007700:Kinetics; D009005:Monosaccharides / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D013379:Substrate Specificity; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000378:metabolism*
D006601:Hexoses; D009005:Monosaccharides; D014534:UDPglucose 4-Epimerase; C030192:tagatose; D005690:Galactose