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N‐Methylation of the Amide Bond by Methyltransferase Asm10 in Ansamitocin Biosynthesis
Author(s) -
Wu Yingying,
Kang Qianjin,
Shang Guangdong,
Spiteller Peter,
Carroll Brian,
Yu TinWein,
Su Wenjin,
Bai Linquan,
Floss Heinz G.
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100062
Subject(s) - methyltransferase , methylation , chemistry , stereochemistry , biosynthesis , amide , complementation , substrate (aquarium) , mutagenesis , o methyltransferase , enzyme , biochemistry , combinatorial chemistry , biology , gene , mutation , ecology , phenotype
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.