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) from Homo sapiens can 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 from Candida tenuis via reduction 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 an in situ oxygen supply through periodic external feed of H2O2 during 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.

Enzyme Module Systems for the Synthesis of Uridine 5′‐Diphospho‐α‐D‐glucuronic Acid and Non‐Sulfated Human Natural Killer Cell‐1 (HNK‐1) Epitope

“…Further enzyme kinetic studies showed no inhibition by UDP‐GlcA and UDP (Figure S6, Supporting Information). Remarkably, inhibition data of human UGDH by the reduced by‐product NADH emphasize the need for an efficient cofactor regeneration of NAD + 23. In summary, these properties reveal His 6 UGDH as a favorable enzyme for the combination with other enzyme modules.…”

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

“…Most importantly, also at a high NAD + concentration being equimolar to UDP‐Glc NOX is necessary to maintain the NADH concentration below inhibitory concentrations (Supporting information, Fig. S8) . This is demonstrated by monitoring substrate and product concentrations during the course of reaction by MP‐CE (Supporting information, Fig.…”

Recent advances in the enzymatic synthesis of sugar-nucleotides using nucleotidylyltransferases and glycosyltransferases

“…In 2014, Nidetzky reported an in vitro one-pot synthesis of UDP-Xyl using UPD-Glc as a donor substrate via an oxidative decarboxylation process (Scheme 18). 48 A two-step conversion of UDP-Glc to UDP-Xyl via UDP-GlcUA was catalysed by UDP-glucose dehydrogenase (hUGDH) and UDP-xylose synthase (hUXS). hUGDH was coupled to a NAD + coenzyme regeneration cascade using a xylose reductase (CtXR) and bovine liver catalase.…”