Tandem Action of Glycosyltransferases in the Maturation of Vancomycin and Teicoplanin Aglycones: Novel Glycopeptides
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- 作者：Heather C. Losey ; Mark W. Peczuh ; Zhong Chen ; Ulrike S. Eggert ; Steven D. Dong ; Istvan Pelczer ; Daniel Kahne ; and Christopher T. Walsh
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：April 17, 2001
- 年：2001
- 卷：40
- 期：15
- 页码：4745 - 4755
- 全文大小：141K
- 年卷期：v.40,no.15(April 17, 2001)
- ISSN：1520-4995
文摘
The glycopeptides vancomycin and teicoplanin are clinically important antibiotics. Thecarbohydrate portions of these molecules affect biological activity, and there is great interest in developingefficient 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, eachglycosyltransferase (Gtf) was characterized for activity either with the aglycones (GtfB, E) or theglucosylated derivatives (GtfC, D) of vancomycin and teicoplanin. GtfB efficiently glucosylates vancomycinaglycone using UDP-glucose as the glycosyl donor to form desvancosaminyl-vancomycin (vancomycinpseudoaglycone), with kcat of 17 min-1, but has very low glucosylation activity, 
le.gif"> 0.3 min-1, for analternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating bothits natural substrate, vancomycin aglycone (kcat = 60 min-1), and an unnatural substrate, teicoplanin aglycone(kcat = 20 min-1). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis ofUDP-
le">-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC andGtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplaninscaffold, 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 resultsestablish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate howthey can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class ofglycopeptide antibiotics.
le.gif"> 0.3 min-1, for analternate substrate, teicoplanin aglycone. In contrast, GtfE is much more efficient at glucosylating bothits natural substrate, vancomycin aglycone (kcat = 60 min-1), and an unnatural substrate, teicoplanin aglycone(kcat = 20 min-1). To test the addition of the 4-epi-vancosamine moiety by GtfC and GtfD, synthesis ofUDP-le">-L-4-epi-vancosamine was undertaken. This NDP-sugar served as a substrate for both GtfC andGtfD in the presence of vancomycin pseudoaglycone (GtfC and GtfD) or the glucosylated teicoplaninscaffold, 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 resultsestablish the enzymatic activity of these four Gtfs, begin to probe substrate specificity, and illustrate howthey can be utilized to make variant sugar forms of both the vancomycin and the teicoplanin class ofglycopeptide antibiotics.