Glycosyltransferases involved in the biosynthesis of glycopeptide antibiotics.

摘要

The glycopeptides vancomycin and teicoplanin are clinically important antibiotics for treating infections caused by Gram-positive pathogens. The rise in vancomycin resistance has created an urgent need for compounds active against resistant bacterial strains. The carbohydrate portions of these molecules affect biological activity, and there is great interest in developing efficient strategies to make carbohydrate derivatives.; Glycosyltransferase genes were cloned from glycopeptide-producing strains of chloroeremomycin (GtfA, GtfB, GtfC), vancomycin (GtfD, GtfE), and balhimycin (bGtfA). The purified glycosyltransferases were then characterized for activity. GtfB and GtfE transferred glucose from UDP-glucose to the natural acceptor substrate, vancomycin aglycone, and GtfE also recognized the teicoplanin aglycone. GtfC and GtfD transferred 4-epi-vancosamine from UDP-4- epi-vancosamine to the vancomycin pseudoaglycone making epivancomycin. In tandem, GtfE and GtfD synthesized a novel hybrid teicoplanin/vancomycin glycopeptide. bGtfA transferred 4-epi-vancosamine to vancomycin pseudoaglycone to make an analog of balhimycin. These results establish enzymatic activity of these five glycosyltransferases.; Understanding the requirements for recognition and catalysis of glycopeptide glycosyltransferases is crucial for combinatorial biosynthesis. The X-ray crystal structure of the glucosyltransferase GtfB has been determined at 1.8 Å. GtfB has a two domain structure with a deep interdomain cleft that is the probable binding site for UDP-glucose. A hydrophobic patch on the surface of the N-terminal domain is proposed to bind the aglycone substrate. Identified through mutagenesis, Asp332 is the best candidate for the general base in the glucosyltransfer reaction. Two structures of GtfA at 3.0 Å have also been determined either in complex with vancomycin or UDP, demonstrating that our predictions for substrate binding are correct.; We generated a library of NDP-glucose derivatives to test the substrate flexibility of GtfE. All four regioisomers of TDP-deoxyglucose and NDP-aminoglucose were recognized and transferred by GtfE to both the vancomycin and teicoplanin scaffolds. Subsequent elaboration with 4-epi-vancosamine by GtfD generated vancomycin and teicoplanin derivatives with variant disaccharides. After transfer of 2-amino-glucose to teicoplanin and vancomycin aglycones by GtfE, N-acylation yielded analogs of vancomycin and teicoplanin active against VanB-type vancomycin-resistant enterococci.; Taken together, the above results demonstrate that the glycopeptide glycosyltransferases will be useful for making many derivatives of glycopeptide antibiotics with altered glycosylation patterns.