title: N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis.
abstract: Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The protein encoded by asm10 belongs to the SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is a 33.0 kDa monomer, as determined by gel filtration. By using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH for Asm10 catalysis were determined to be 32 °C and 10.0, respectively. Asm10 also showed broad substrate flexibility toward other N-desmethyl-ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis, possibly through the binding of SAM. The characterization of this unique N-methyltransferase has enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds; this will allow known potential drugs to be modified.
authors.affiliation: State Key Laboratory of Stress Cell Biology and School of Life Sciences, Xiamen University, Xiamen 361005, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Wu
authors.forename: Qianjin
authors.initials: Q
authors.lastname: Kang
authors.forename: Guangdong
authors.initials: G
authors.lastname: Shang
authors.forename: Peter
authors.initials: P
authors.lastname: Spiteller
authors.forename: Brian
authors.initials: B
authors.lastname: Carroll
authors.forename: Tin-Wein
authors.initials: TW
authors.lastname: Yu
authors.forename: Wenjin
authors.initials: W
authors.lastname: Su
authors.forename: Linquan
authors.initials: L
authors.lastname: Bai
authors.forename: Heinz G
authors.initials: HG
authors.lastname: Floss
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000378:metabolism; D000577:Amides / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000737:chemistry / Q000378:metabolism; D008453:Maytansine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000577:Amides; D047029:Lactams, Macrocyclic; D008453:Maytansine; C015682:ansamitocins; D008780:Methyltransferases