The in vitro Characterization of Polyene Glycosyltransferases AmphDI and NysDI

Abstract The overproduction, purification, and in vitro characterization of the polyene glycosyltransferases (GTs) AmphDI and NysDI are reported. A novel nucleotidyltransferase mutant (RmlA Q83D) for the chemoenzymatic synthesis of unnatural GDP–sugar donors in conjunction with polyene GT‐catalyzed sugar exchange/reverse reactions allowed the donor and acceptor specificities of these novel enzymes to be probed. The evaluation of polyene GT aglycon and GDP–sugar donor specificity revealed some tolerance to aglycon structural diversity, but stringent sugar specificity, and culminated in new polyene analogues in which L ‐gulose or D ‐mannose replace the native sugar D ‐mycosamine.