NDP-sugar glycosyltransferases are critical for the biosynthesis of all complex carbohydrates and glycoconjugates. This large and diverse class of enzymes catalyzes the transfer of saccharide units onto the target compounds to create a diverse set of macromolecules in plants, animals, and bacteria: glycolipids, lipopolysaccharides, the glycan structures in plants, glycoproteins, and glycosylated natural products of biomedical importance like antibiotics, hormones, antitumor agents, and cardiac glycosides. Initially, we intend to determine the X-ray crystal structures of 3 subfamilies of the family 1 NDP-sugar glycosyltransferases (GTFs): GTFs involved in the biosynthesis of vancomycin group antibiotics, GTFs which create sterol glucosides, and two GTFs involved in the glycosylation of diacylglycerol. Our goals are to elucidate (1) the physical basis for the recognition of NDP-sugars, (2) the physical basis for the recognition of the aglycone acceptors, and (3) the mechanism of glycosyltransfer. Understanding the structural diversity of these enzymes will enhance our understanding of glycobiology, particularly regarding the biosynthesis of glycolipids, glycosteroids, and antibiotics. A better understanding of the glycosylation of secondary metabolites will also open up new avenues for antibiotic design against pathogenic organisms and the design of other biomedically relevant compounds (e.g., cardiac glycosides or antitumor agents). Family 1 NDP-sugar glycosyltransferases show significant conservation of functionality among homologous enzymes across species as well as between analogous enzymes within a species, despite very low levels of amino acid sequence conservation (often <25% identity) in many cases. Recent research has also detected distinct elements of structural homology that are conserved between all members of this functional class. With comparative protein sequence analysis, a structural database of the family 1 enzyme could allow the modeling of any structurally unknown protein within this family. Moreover, the relatively simple bi-domain design of family 1 NDP-sugar glycosyltransferases raises the possibility of designing chimeric enzymes with novel functionalities. Mixing hybrid enzyme design with combinatorial biosynthesis could provide practical ways to produce new glycoconjugates of biomedical importance.
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