UDP-Glc-4-epimerase

We have developed a novel enzymatic reaction cycle for the synthesis of LacNAc with in situ regeneration of UDP-Glc (25) and UDP-Gal (16) (Fig. 27) [273, 275]. The unique character of sucrose synthase to generate 25 by the cleavage of sucrose (24) with UDP makes this enzyme more favourable compared with pyrophosphorylases. In this way UDP-Glc is directly regenerated and further epimerized to 16 by UDP-Glc 4-epimerase from yeast, yielding the donor substrate for human milk [11-4 GalT. In comparison to other published... 

Fig. 28. Reaction mechanism of UDP-Glc 4-epimerase (EC 5.1.3.2). E-NAD enzyme-bound cofactor...

Reactivation of UDP-Glc 4-epimerase can be achieved by incubation with the transition state analogs, dUDP- or dTDP-6-deoxy-4-ketoglucose (40, 37), resulting in an enzyme-bound cofactor regeneration (Fig. 30). The full activity of UDP-Glc 4-epimerase was recovered and the stability of the enzyme increased by incubation with these transition state analogs which have been synthesized by us on a 0.1 to 1 g scale [272,331,332] (see also Sections 6.1 and 6.2). With this... 

Table 6. Reductive Inactivation of UDP-Glc 4-epimerase by different monosaccharides and derivatives thereof [275]...

Polyphosphate kinase has been found able to phosphorylate nucleoside diphosphates to give nucleoside triphosphates, using PolyP as a phosphate donor. Therefore, the possibility of using PolyP and polyphosphate kinase instead of phosphoenol pyruvate and pyruvate kinase for enzymatic oligosaccharide synthesis was examined, because PolyP is quite cheap when compared with phosphoenol pyruvate (Noguchi and Shiba, 1998 Shiba et al, 2000). Attempts were made to synthesize N-acetyllactosamine (Gal (f> 1-4) GlcNAc) using the nucleoside diphosphate kinase-like activity of polyphosphate kinase, where UDP-Glc pyrophosphorylase and UDP-Glc 4-epimerase catalyse the synthesis of UDP-Glc from... 

Figure 4 Enzymatic synthesis of Galal,3Galpl,4GlcNAcpOR with in situ UDP-Gal regeneration. Four enzymes are used in the synthesis sucrose synthase (SusA), UDP-Glc 4 -epimerase, pl,4GalT, and a1,3GalT.

Starting from UDP-Glc, a variety of a-D-Gal-(l- 4)-P-D-Gal derivatives have been prepared using a UDP-Glc-4-epimerase-a-l,3-Gal-transferase fusion enzyme. The fusion protein enhanced reaction rates by 300yo compared to the same reactions carried out with equivalent amounts of the two separate en-zymes. A one-pot route to ketoses, such as 38, from glycerol and pyrophosphate, via their phosphorylated precursors 37 has been developed using phytase (Scheme 8). ... 

Following the known biosynthetic pathway shown in Scheme 1, UDP-Glc can be regenerated by sucrose synthase and applied into glycosylation. On the other hand, UDP-Gal regeneration system was established in two approaches based on two different biosynthetic pathways. By adding UDP-Glc-4-epimerase (GalE, EC 5.1.3.2) into the sucrose synthase-catalyzed UDP-Glc regeneration... 

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