Enzymatic synthesis of UDP-GlcN by a two step hollow fiber enzyme reactor system.
UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.
Ropp
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Department of Pediatrics, University of North Carolina, Chapel Hill 27599-7220.
Cheng
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D000117:Acetylglucosamine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005944:Glucosamine / Q000031:analogs & derivatives; D006593:Hexokinase / Q000378:metabolism; D010733:Phosphoglucomutase / Q000378:metabolism; D013379:Substrate Specificity; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000037:antagonists & inhibitors; D014532:Uridine Diphosphate Glucose / Q000138:chemical synthesis*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000138:chemical synthesis; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis*; D014544:Uridine Triphosphate / Q000378:metabolism
D014539:Uridine Diphosphate Sugars; C026712:UDP-glucosamine; D014537:Uridine Diphosphate N-Acetylglucosamine; D006593:Hexokinase; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D010733:Phosphoglucomutase; D005944:Glucosamine; D014544:Uridine Triphosphate; D014532:Uridine Diphosphate Glucose; D000117:Acetylglucosamine