RESUMO
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 ß4-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)-ß-l-fucose by the bifunctional l-fucokinase/GDP-ß-l-Fuc pyrophosphorylase of Bacteroides fragilis 9343.
Assuntos
Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Trissacarídeos/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/genética , Bacteroides fragilis/metabolismo , Catálise , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fucosiltransferases/isolamento & purificação , Regulação Enzimológica da Expressão Gênica , Guanosina Difosfato Fucose/metabolismo , Lactose/metabolismo , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
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.