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Complete Biosynthetic Pathway of Alazopeptin, a Tripeptide Consisting of Two Molecules of 6‐Diazo‐5‐oxo‐ l ‐norleucine and One Molecule of Alanine
Author(s) -
Kawai Seiji,
Sugaya Yuko,
Hagihara Ryota,
Tomita Hiroya,
Katsuyama Yohei,
Ohnishi Yasuo
Publication year - 2021
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202100462
Subject(s) - tripeptide , chemistry , norleucine , diazo , enzyme , biochemistry , biosynthesis , stereochemistry , dehydroalanine , amino acid , heterologous , gene , methionine , organic chemistry
DON (6‐diazo‐5‐oxo‐ l ‐norleucine), a diazo‐containing amino acid, has been studied for more than 60 years as a potent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala‐DON‐DON, which has antitumor activity, by gene inactivation and in vitro analysis of recombinant enzymes. We also established heterologous production of N ‐acetyl‐DON in Streptomyces albus . DON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly, transmembrane protein AzpL was indicated to catalyze diazotization using 5‐oxolysine and nitrous acid as substrates. Site‐directed mutagenesis of AzpL indicated that the hydroxy group of Tyr‐93 is important for the diazotization. These findings expand our knowledge of the enzymology of N−N bond formation.