Specific enrichment of nonribosomal peptide synthetase module by an affinity probe for adenylation domains |
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| Affiliation: | 1. Department of Pharmaceutical Technology, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 50005 Hradec Králové, Czech Republic;2. Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic;3. Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 50005 Hradec Králové, Czech Republic;4. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 50005 Hradec Králové, Czech Republic;5. Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava, Partyzánské náměstí 7, 70200 Ostrava, Czech Republic;1. Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9038, USA;2. Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390-9041, USA;3. Scynexis, Inc. (now Avista Pharma Solutions), 3501 Tricenter Blvd, Suite C, Durham, NC 27713, USA;1. Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China;2. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China |
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| Abstract: | We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5′-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated l-Phe-AMS (l-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates l-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes. |
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| Keywords: | Nonribosomal peptide synthetase Polyketide synthase Adenylation domain Affinity probe Gramicidin S |
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