title: Characterization of UDP-glucose 4-epimerase from Pyrococcus horikoshii: regeneration of UDP to produce UDP-galactose using two-enzyme system with trehalose.
abstract: A gene encoding a putative UDP-glucose 4-epimerase (pGALE) in Pyrococcus horikoshii was cloned and expressed in Escherichia coli. The purified enzyme could reversibly catalyze both the synthesis of UDP-Gal and UDP-Glc but preferred the binding of UDP-Gal by approximately 10-fold. The optimum pH and temperature were 6.5 and 65°C. The enzyme acted effectively without the addition of nicotinamide adenine dinucleotide (NAD(+)), possibly due to the presence of tightly bound NAD(+). In particular, pGALE could be coupled with trehalose synthase (TreT) from P. horikoshii to regenerate UDP-Gal from UDP. The possible byproduct of glycosyltransferase, UDP, was capable of being converted to UDP-Glc with trehalose by TreT, and UDP-Glc was simultaneously converted to UDP-Gal by pGALE. Conclusively, the results suggest that pGALE and TreT with trehalose is an effective one-pot two-enzyme system for the regeneration of UDP-Gal, a high-cost substrate of galactosyltransferase, to complete a sugar nucleotide cycle.
authors.affiliation: Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Seung-Kyung
authors.initials: SK
authors.lastname: Chung
authors.forename: Soo-In
authors.initials: SI
authors.lastname: Ryu
authors.forename: Soo-Bok
authors.initials: SB
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014199:Trehalose / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014531:Uridine Diphosphate Galactose; D014530:Uridine Diphosphate; D014199:Trehalose; D014534:UDPglucose 4-Epimerase