Amide N-glycosylation by Asm25, an N-glycosyltransferase of ansamitocins.
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 beta-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.
Zhao
Peiji
P
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China.
Bai
Linquan
L
Ma
Juan
J
Zeng
Ying
Y
Li
Lei
L
Zhang
Yirong
Y
Lu
Chunhua
C
Dai
Huanqin
H
Wu
Zhaoxian
Z
Li
Yaoyao
Y
Wu
Xuan
X
Chen
Gang
G
Hao
Xiaojiang
X
Shen
Yuemao
Y
Deng
Zixin
Z
Floss
Heinz G
HG
D000192:Actinomycetales / Q000201:enzymology; D000577:Amides / Q000378:metabolism*; D000935:Antifungal Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001487:Basidiomycota / Q000187:drug effects; D045744:Cell Line, Tumor; D005964:Glucosyltransferases / Q000378:metabolism*; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D007769:Lactams / Q000737:chemistry / Q000378:metabolism; D008453:Maytansine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D020673:Protein Renaturation; D013379:Substrate Specificity; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D000577:Amides; D000935:Antifungal Agents; D000970:Antineoplastic Agents; D001426:Bacterial Proteins; D006027:Glycosides; D007769:Lactams; D008453:Maytansine; C015682:ansamitocins; D005964:Glucosyltransferases; C410788:N-glycosyltransferase, Nocardia aerocolonigenes; D014532:Uridine Diphosphate Glucose