Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: Novel glycopeptides

The glycopeptides vancomycin and teicoplanin are clinically important antib iotics. The carbohydrate portions of these molecules affect biological acti vity and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransfe rases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escher ichia coli. After expression and purification, each glycosyltransferase (Gt f) was characterized for activity either with the aglycones (GtfB, E) or th e glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB ef ficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosy l donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), wit h k(cat) of 17 min(-1), but has very low glucosylation activity, less than or equal to 0.3 min(-1), for an alternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating both its natural substrate, vancomycin aglycone (k(cat) = 60 min(-1)), and an unnatural subs trate, teicoplanin aglycone (k(cat) = 20 min(-1)). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis of UDP-beta -L-4-e pi-vancosamine was undertaken. This NDP-sugar served as a substrate for bot h GtfC and GtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD ) or the glucosylated teicoplanin scaffold, 7 (GtfD). The GtfC product was the 4-epi-vancosaminyl form of vancomycin. Remarkably, GtfD was able to uti lize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor , UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomyci n glycopeptide. These results establish the enzymatic activity of these fou r Gtfs, begin to probe substrate specificity, and illustrate how they can b e utilized to make variant sugar forms of both the vancomycin and the teico planin class of glycopeptide antibiotics.