Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains

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 affinity probe for A domains that could be used to facilitate the specific isolation and identification 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.
Source: Springer protocols feed by Protein Science - Category: Biochemistry Source Type: news