Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis ofN‐Acetyllactosamine

Abstract

In this study, we describe the direct synthesis of uridine 5'-diphosphate galactose (UDP-Gal) by a wild-type bacterial thymidylyltransferase (RmlA), which is used to synthesize thymidine 5'-diphosphate glucose (TDP-glucose) in nature. By using magnesium (Mg 2+ ) as a cofactor and a reaction temperature of 55 8C, a one hundred milligram-scale synthesis of UDP-Gal was achieved by RmlA. In addition, RmlA was site-specifically and covalently immobilized on magnetic nanoparticles (MNPa) The resulting RmlA-MNP complex retained almost 95% of its activity after reuse in ten consecutive enzyme assays. Furthermore, b-1,4-galactosyltransferase (GalT) from Neisseria meningitides was successfully overexpressed and purified by using an intein-mediated protein expression system. GalT was relatively stable at 25 8C, and its activity was enhanced in the presence of DTT and BSA. Thus, it was feasible to synthesize N-acetyllactosamine (LacNAc) using RmlA and GalT in a sequential addition of enzyme and adjustment of thereaction temperature. These results demonstrate the potential applications of bacterial RmlA in carbohydrate synthesis.

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

“…In the first pot, the synthesis of UDP‐Gal was achieved using optimal conditions, including 20 mM Gal ( 3a ), 20 mM ATP, and 40 mM MgCl 2 in 100 mM Tris‐HCl buffer (pH 9.0) in the presence of MtGalK and RmlA at concentrations of 2 μg mL −1 and 500 μg mL −1 , respectively. The reaction mixture was stirred at 55 °C for 4 h. The production of UDP‐Gal was monitored by RP‐HPLC with a UV detector as described previously 19. When the formation of UDP‐Gal reached a 60% yield, the reaction mixture was cooled to 37 °C followed by the addition of Lac acceptor 5 (10 mM) and LgtC (100 μg mL −1 ) to the solution and further incubation for another 3 h. The trisaccharide was purified by size‐exclusion chromatography to yield 1b in a 91% yield (960 mg).…”

“…The advantages of using thermophilic enzymes in organic synthesis at elevated temperatures include high reaction rates due to the decreased viscosity and increased diffusion of substrates and products and the improved bioavailability and solubility of organic compounds 18. Recently, we developed a system using the thermophilic enzyme RmlA (glucose‐1‐phosphate thymidylyltransferase, EC 2.7.7.24, from Aneurinibacillus thermoaerophilus DSM10155) to smoothly convert Gal‐1‐P to UDP‐Gal at 55 °C, providing a practical strategy for the synthesis of UDP‐Gal on the hundreds‐of‐milligrams scale 19…”

Enzymatic Synthesis of Human Milk Fucosides α1,2‐Fucosyl para‐Lacto‐N‐Hexaose and its Isomeric Derivatives

“…The sugar donors required for the GT‐catalyzed reaction, uridine diphosphate galactose (UDP‐Gal) and uridine diphosphate N ‐acetyl glucosamine (UDP‐GlcNAc), were generated through sequential one‐pot enzymatic reaction with slight modification . UDP‐GlcNAc was prepared by the action of N ‐acetyl hexosamine kinase from Bifidobacterium longum (NahK) and glucose‐1‐phosphate thymidylyltransferase from Aneurinibacillus thermoaerophilus (RmlA); in addition, UDP‐Gal was generated using galactokinase from Meiothermus taiwanensis (MtGalK) and UDP‐sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP) . By combining HP1105 and WbgO with the corresponding UDP‐sugar‐generating enzymes in the sequential one‐pot system, lactose 1 was smoothly converted into defined lengths of HMO backbone containing lacto‐ N ‐biose (type‐1 LacNAc) repeats ( 2 – 5 ), as shown in Scheme .…”

Water‐Soluble Sulfo‐Fluorous Affinity (SOFA) Tag‐Assisted Enzymatic Synthesis of Oligosaccharides

“…In the proof‐of‐concept experiment, we examined two SOFA‐tagged acceptors ( 1 a and 1 c ) and one conventional azidohexyl‐tagged acceptor ( 1 b ). SOFA‐tagged LacNAc 14 a was prepared from GlcNAc 1 a using β‐1,4‐galactosyltransferase from Neisseria meningitidis (NmGalT) in the presence of uridine diphosphate galactose (UDP‐Gal), which was generated by a sequential one‐pot enzymatic reaction using galactokinase from Meiothermus taiwanensis (MtGalK) and glucose‐1‐phosphate thymidylyltransferase from Aneurinibacillus thermoaerophilus (RmlA) as reported previously with minor modifications (Scheme ). After incubation at 25 °C for 6 h, the reaction mixture was transferred to an FSPE column and eluted with water followed by methanol.…”

“…Therefore, we used aliphatic SOFA‐tagged substrates (compound number with a ) for further neutral oligosaccharide syntheses. As shown in Scheme , lactoside 2 a was galactosylated by LgtC, an α‐1,4‐galactosyltransferase from N. meningitidis , in the presence of UDP‐Gal with 2 h of incubation at 37 °C by a sequential one‐pot enzymatic synthesis procedure to give Gb3 18 in 83% yield (19 mg). The efficiency of LgtC catalysis toward SOFA‐tagged lactose 2 a was similar to that of azidohexyl lactose 2 b that we reported previously (91%) …”