Affinity purification method for the identification of nonribosomal peptide biosynthetic enzymes using a synthetic probe for adenylation domains

Abstract

A series of inhibitors have been designed based on 5′- O -sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affi nity probe for A domains that could be used to facilitate the specifi c isolation and identifi cation of NRPS modules. Our synthetic probe, which is a biotinylated variant of L -Phe-AMS (L -Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L -Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L -Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.