Publications

• Crystal structure of vancosaminyltransferase GtfD from the vancomycin biosynthetic pathway: interactions with acceptor and nucleotide ligands.

  Mulichak A.M., Lu W., Losey H.C., Walsh C.T., Garavito R.M.

  The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that ... >> More

  The TDP-vancosaminyltransferase GtfD catalyzes the attachment of L-vancosamine to a monoglucosylated heptapeptide intermediate during the final stage of vancomycin biosynthesis. Glycosyltransferases from this and similar antibiotic pathways are potential tools for the design of new compounds that are effective against vancomycin resistant bacterial strains. We have determined the X-ray crystal structure of GtfD as a complex with TDP and the natural glycopeptide substrate at 2.0 A resolution. GtfD, a member of the bidomain GT-B glycosyltransferase superfamily, binds TDP in the interdomain cleft, while the aglycone acceptor binds in a deep crevice in the N-terminal domain. However, the two domains are more interdependent in terms of substrate binding and overall structure than was evident in the structures of closely related glycosyltransferases GtfA and GtfB. Structural and kinetic analyses support the identification of Asp13 as a catalytic general base, with a possible secondary role for Thr10. Several residues have also been identified as being involved in donor sugar binding and recognition. << Less

  Biochemistry 43:5170-5180(2004) [PubMed] [EuropePMC]

• Tandem action of glycosyltransferases in the maturation of vancomycin and teicoplanin aglycones: novel glycopeptides.

  Losey H.C., Peczuh M.W., Chen Z., Eggert U.S., Dong S.D., Pelczer I., Kahne D., Walsh C.T.

  The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltran ... >> More

  The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives. To this end, genes encoding four glycosyltransferases, GtfB, C, D, E, were subcloned from Amycolatopsis orientalis strains that produce chloroeremomycin (GtfB, C) or vancomycin (GtfD, E) into Escherichia coli. After expression and purification, each glycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or the glucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycin aglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycin pseudoaglycone), with k(cat) of 17 min(-1), but has very low glucosylation activity, < or = 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 substrate, 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-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both 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 utilize both an unnatural acceptor, 7, and an unnatural nucleotide sugar donor, UDP-4-epi-vancosamine, to synthesize a novel hybrid teicoplanin/vancomycin glycopeptide. These results establish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate how they can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class of glycopeptide antibiotics. << Less

  Biochemistry 40:4745-4755(2001) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.