Structure-function relationships in eukaryotic and prokaryotic family 6 glycosyltransferases.

摘要

Carbohydrate Active Enzyme family 6 (CA6) glycosyltransferases (GTs) are type II transmembrane proteins localized in the Golgi apparatus. CA6 GTs have a GT-A fold, a type of structure that resembles the Rossman fold and catalyze the transfer either galactose (Gal) or N-acetylgalactosamine (GalNAc) from the UDP nucleotide sugar to an non-reducing terminal Gal or GalNAc on an acceptor via an alpha-1,3 linkage. In this reaction, the anomeric configuration of the sugar moiety of the donor is retained in the product. CA6 GTs includes the histo-blood group A and B GTs, alpha-galactosyltransferase (alpha3GT), Forssman glycolipid synthase (FS), isogloboside 3 synthase (iGb3) in mammals. alpha3GT and its products (alpha-Gal epitode) are present in most mammals but are absent in humans and old world primates because of inactivating mutations. The absence of alpha3GT and its products results in the production of anti-alpha-Gal epitope natural antibodies in these species. Up to date, the catalytic mechanisms of the CA6 GTs are not well understood. Based on previous structural and mutagenesis studies of bovine alpha3GT, we investigated active site residues (His315, Asp316, Ser318, His319, and Lys359) that are highly conserved among CA6 GTs. We have also investigated the role of the C-terminal region by progressive C-terminal truncations. Findings from these studies clarify the functional roles of these residues in structure, catalysis, and specificity in these enzymes and have implications for their catalytic mechanisms.;GTs are useful tools in synthesis of glycans for various applications in science and medicine. Methods for the large scale production of pure glycans are continuously being developed. We created a limited randomized combinatorial library based on knowledge of structural information and sequence analysis of the enzyme and its mammalian homologues. Two GalNAc-specific variants were identified from the library and one Glc-specific variant was identified by site-direct mutagenesis. The glycosyltransferase activities of these variants are expected to be improved by further screens of libraries which are designed using the variants as templates.;The mammalian CA6 GTs that have been characterized to date are metal-dependent and require the divalent cation, Mn2+ for activity. In some recently-discovered bacterial CA6 GTs, the DXD sequence that is present in eukaryotic GTs is replaced by NXN. We cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that has been linked with inflammatory bowel disease. Functional characterization shows it is a metal-independent monomeric GT that efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A glycan. Mutational studies indicated that despite the lack of a metal cofactor there are similarities in structure-function relationships between the bacterial and vertebrate family 6 GTs.