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Novel Activity of UDP-Galactose-4-Epimerase for Free Monosaccharide and Activity Improvement by Active Site-Saturation Mutagenesis

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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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Acknowledgments

This work was supported by the Korean Ministry of Education, Science, and Technology (R01-2009-0070677, R1-2007-000-202310). P. Kim was further supported by a 2009 Research Grant from the Catholic University of Korea.

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Authors and Affiliations

  1. Department of Biotechnology, Catholic University of Korea, Bucheon, Gyeonggi, 420-743, South Korea

    Hye-Jung Kim, Sueng Yeun Kang & Pil Kim

  2. Food & Bio Research Center, Samyang Genex Co., Yuseong, Daejon, 305-717, South Korea

    Jong Jin Park

Authors
  1. Hye-Jung Kim
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  2. Sueng Yeun Kang
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  3. Jong Jin Park
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  4. Pil Kim
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Corresponding author

Correspondence to Pil Kim.

Electronic Supplementary Material

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Figure S1

Optimal conditions (pH, temperature, and [NAD+]) for 4-epimerization of glucose (PDF 23 kb)

Figure S2

SDS-PAGE of GalE wild-type and variant N179S (soluble fraction) (PDF 17 kb)

Figure S3

Chromatograms of GalE-mediated conversion of glucose into galactose (a), fructose into tagatose (b), and psicose into sorbose (c). Chromatograms obtained after 4 h were superimposed on chromatograms obtained before the reaction (PDF 99 kb)

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Kim, HJ., Kang, S.Y., Park, J.J. et al. Novel Activity of UDP-Galactose-4-Epimerase for Free Monosaccharide and Activity Improvement by Active Site-Saturation Mutagenesis. Appl Biochem Biotechnol 163, 444–451 (2011). https://doi.org/10.1007/s12010-010-9052-7

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  • DOI: https://doi.org/10.1007/s12010-010-9052-7

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