title: NahK/GlmU fusion enzyme: characterization and one-step enzymatic synthesis of UDP-N-acetylglucosamine.
abstract: The availability of uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a prerequisite for the GlcNAc-transferase-catalyzed glycosylation reaction. UDP-GlcNAc has already been synthesized using an N-acetylhexosamine 1-kinase (NahK) and a GlcNAc-1-P uridyltransferase (truncated GlmU) and here, a fusion enzyme was constructed with truncated GlmU and NahK. After determination of the optimum catalytic condition (pH 8.0 at 40 °C), the fusion enzyme was used to synthesize UDP-GlcNAc in a single step with a yield of 88 % from GlcNAc, ATP and UTP. Furthermore, a simplified purification method was demonstrated using separation by gel filtration after by-product digestion with alkaline phosphatase. An overall yield of 77 % and a purity of over 90 % were achieved.
authors.affiliation: National Glycoengineering Research Center, Shandong University, B604 Luneng Keji Dasha, 29-1 Shanda Nanlu, Jinan, 250100 Shandong, People's Republic of China.
authors.forename: Yafei
authors.initials: Y
authors.lastname: Zhai
authors.forename: Min
authors.initials: M
authors.lastname: Liang
authors.forename: Junqiang
authors.initials: J
authors.lastname: Fang
authors.forename: Xiaoyue
authors.initials: X
authors.lastname: Wang
authors.forename: Wanyi
authors.initials: W
authors.lastname: Guan
authors.forename: Xian-Wei
authors.initials: XW
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Fengshan
authors.initials: F
authors.lastname: Wang
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods; D004795:Enzyme Stability; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000096:biosynthesis* / Q000302:isolation & purification; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D004798:Enzymes; D011993:Recombinant Fusion Proteins; D014537:Uridine Diphosphate N-Acetylglucosamine; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate; D000117:Acetylglucosamine