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Published on: November 23, 2016
Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic
Fumihiro Ishikawa1, Hideaki Kakeya1
1Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Science, Kyoto University, Sakyo-ku, Kyoto, 606-8501, Japan.
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 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.
