Amide N-glycosylation by Asm25, an N-glycosyltransferase of ansamitocins

Abstract

Ansamitocins are potent antitumor maytansinoids produced by Actinosynnema pretiosum. Their biosynthesis involves the initial assembly of a macrolactam polyketide, followed by a series of postpolyketide synthase (PKS) modifications. Three ansamitocin glycosides were isolated from A. pretiosum and fully characterized structurally as novel ansamitocin derivatives, carrying a (3-D-glucosyl group attached to the macrolactam amide nitrogen in place of the N-methyl group. By gene inactivation and complementation, asm25 was identified as the N-glycosyltransferase gene responsible for the macrolactam amide N-glycosylation of ansamitocins. Soluble, enzymatically active Asm25 protein was obtained from asm25-expressing E, coli by solubilization from inclusion bodies. Its optimal reaction conditions, including temperature, pH, metal ion requirement, and Km/Kcat, were determined. Asm25 also showed broad substrate specificity toward other ansamycins and synthetic indolin-2-ones. To the best of our knowledge, this represents the first in vitro characterization of a purified antibiotic N-glycosyltransferase.