title: Enzymatic Redox Cascade for One-Pot Synthesis of Uridine 5'-Diphosphate Xylose from Uridine 5'-Diphosphate Glucose.
abstract: Synthetic ways towards uridine 5'-diphosphate (UDP)-xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP-glucose 6-dehydrogenase (hUGDH) and UDP-xylose synthase 1 (hUXS) fromHomo sapienscan be used for the efficient production of pure UDP-α-xylose from UDP-glucose. In a mimic of the natural biosynthetic route, UDP-glucose is converted to UDP-glucuronic acid by hUGDH, followed by subsequent formation of UDP-xylose by hUXS. The nicotinamide adenine dinucleotide (NAD+) required in the hUGDH reaction is continuously regenerated in a three-step chemo-enzymatic cascade. In the first step, reduced NAD+(NADH) is recycled by xylose reductase fromCandida tenuis viareduction of 9,10-phenanthrenequinone (PQ). Radical chemical re-oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H2O2) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo-enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP-xylose as well as an adequate oxygen supply for PQ re-oxidation as major bottlenecks of effective performance of the overall multi-step reaction system. Net oxidation of UDP-glucose to UDP-xylose by hydrogen peroxide (H2O2) could thus be achieved when using anin situoxygen supply through periodic external feed of H2O2during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g l-1) UDP-α-xylose. After two-step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi-step redox cascades in the synthesis of nucleotide sugar products.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology NAWI Graz, Petersgasse 12/I, A-8010 Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eixelsberger
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology NAWI Graz, Petersgasse 12/I, A-8010 Graz, Austria ; Austrian Centre of Industrial Biotechnology Petersgasse 14, A-8010 Graz, Austria, ; phone: (+43)-316-873-8400 ; e-mail: bernd.nidetzky@tugraz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
keywords: UDP-glucose dehydrogenase; UDP-xylose synthase; biosynthetic cascade; carbohydrates; multi-enzyme catalysis; nucleotide sugars; uridine 5′-diphosphate (UDP)