LAUSR

During the biosynthesis of alazopeptin, a tripeptide composed of two molecules of 6‐diazo‐5‐oxo‐L‐norleucine (DON) and one of alanine, the α/β hydrolase AzpM synthesizes the DON‐DON dipeptide using DON tethered to the carrier protein AzpF (DON‐AzpF). However, whether AzpM catalyzes the condensation of DON‐AzpF with DON or DON‐AzpF remains unclear. Here, to distinguish between these two condensation possibilities, the reaction catalyzed by AzpM was examined in vitro using a DON analogue, azaserine (AZS). We found that AzpM catalyzed the condensation between AZS‐AzpF and DON‐AzpF, but not between AZS‐AzpF and DON. Possible reaction intermediates, DON‐DON‐AzpF and AZS‐AZS‐AzpF, were also detected during AzpM‐catalyzed dipeptide formation from DON‐AzpF and AZS‐AzpF, respectively. From these results, we concluded that AzpM catalyzed the condensation of the two molecules of DON‐AzpF and subsequent hydrolysis to produce DON‐DON. Thus, AzpM is an unprecedented α/β hydrolase that catalyzes dipeptide synthesis from two molecules of a carrier protein‐tethered amino acid.