, ad iazo-containing amino acid, has been studied for more than 60 years as ap otent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala-DON-DON,w hichh as antitumor activity,bygene inactivation and in vitro analysis of recombinant enzymes.W ea lso established heterologous production of N-acetyl-DON in Streptomyces albus.D ON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly,t ransmembrane 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 À Nbond formation.

, ad iazo-containing amino acid, has been studied for more than 60 years as ap otent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala-DON-DON,w hichh as antitumor activity,bygene inactivation and in vitro analysis of recombinant enzymes.W ea lso established heterologous production of N-acetyl-DON in Streptomyces albus.D ON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly,t ransmembrane 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 À Nbond formation.