Fucosyltransferases (FucTs) are essential tools for the synthesis of fucosylated glycoconjugates. Multistep enzyme catalysis of fucosylated glycans is not limited as long as isolated and well-characterized FucTs are available. The present paper introduces a novel bacterial α1,2-FucT of the glycosyltransferase family 11 encoded by the gene wbgL in the E. coli O126 genome, which only displays 25-30% homology to previously published α1,2-FucTs. A tailor made cloning and expression strategy allowed the successful production of active soluble enzyme in the cytoplasm of E. coli BL21(DE3) and E. coli JM109(DE3), respectively. The lack of a DxD motif and its high activity without divalent metal ions suggests that WbgL belongs to the GT-B fold superfamily. Substrate screening revealed the highest activity for β4-linked galactoside acceptor substrates, such as lactose and lactulose, making WbgL unique among other characterized α1,2-FucTs. Based on its excellent kinetic efficiency for lactose, we present here a sequential reaction strategy for the synthesis of α1,2-fucosyllactose in one pot including the synthesis of the donor substrate 3,3'-Diaminobenzidine (GDP)-β-l-fucose by the bifunctional l-fucokinase/GDP-β-l-Fuc pyrophosphorylase of Bacteroides fragilis 9343.
Aryl sulfotransferase IV (AstIV) from rat liver was overexpressed in Escherichia coli and purified to homogeneity. Using the produced mammalian liver enzyme, sulfation-the Phase II conjugation reaction-of optically pure silybin diastereoisomers (silybin A and B) was tested. As a result, silybin B was sulfated yielding 20-O-silybin B sulfate, whereas silybin A was completely resistant to the sulfation reaction. Milligram-scale sulfation of silybin B was optimized employing resting E. coli cells producing AstIV, thus avoiding the use of expensive 3'-phosphoadenosine-5'-phosphate cofactor and laborious enzyme purification. Using this approach, we were able to reach 48 % conversion of silybin B into its 20-sulfate within 24 h. The sulfated product was isolated by solid phase extraction and its structure was characterized by HRMS and NMR. Sulfation reaction of silybin appeared strictly stereoselective; only silybin B was sulfated by AstIV.
Tailor-made strategies for the stereo-and regioselective multi-step enzymatic synthesis of glycoconjugates require well characterized glycosyltransferases andc arbohydrate modifying enzymes. We here report on an ovel enzyme cascade for the synthesiso fu ridine 5'-diphospho-a-d-glucuronic acid (UDP-GlcA) andt he non-sulfated human natural killer cell-1 (HNK-1) epitope including in situ regenerationo fU DP-GlcA andt he cofactor nicotinamide adenine dinucleotide NAD + + by the combination of four enzymes in one-pot.I nt he first enzyme module sucroses ynthase 1( SuSy1) is usedt op roduce uridine 5'-diphospho-a-d-glucose (UDP-Glc) from sucrose and uridine 5'-diphosphate (UDP). Thec ombination with UDP-Glc dehydrogenase in the second enzyme module leads to the synthesiso fU DP-GlcA with concomitant in situ regeneration of the cofactor NAD + + by nicotinamide adenine dinucleotide hydride (NADH)-oxidase.I nt he third enzyme module the mammalian glucuronyltransferase GlcAT-Pc atalyzes the synthesiso ft he non-sulfated HNK-1e pitope by regioselective transfer of GlcA onto N-acetyllactosamine type 2( LacNAc type 2). We present ac omprehensive studyo ns ubstrate kinetics,s ubstrate specificities,v ariation and relation of enzyme activitiesa s well as cross inhibition of intermediate products.With optimized reactionc onditions we obtain superior product yieldsw ith streamlined synthesisc osts for the expensiven ucleotide sugar UDP-GlcA and cofactor NAD + + .
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