title: Expanding Substrate Promiscuity by Engineering a Novel Adenylating-Methylating NRPS Bifunctional Enzyme.
abstract: Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega-enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes.
authors.affiliation: Department of Pharmaceutical Sciences, University of Kentucky, BioPharm Complex (Room 423), 789 South Limestone Street, Lexington, KY, 40536-0596, USA.
authors.forename: Sanjib K
authors.initials: SK
authors.lastname: Shrestha
authors.affiliation: Department of Pharmaceutical Sciences, University of Kentucky, BioPharm Complex (Room 423), 789 South Limestone Street, Lexington, KY, 40536-0596, USA. sylviegtsodikova@uky.edu.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Garneau-Tsodikova
mesh_terms: D000249:Adenosine Monophosphate; D000596:Amino Acids / Q000378:metabolism; D001688:Biological Products / Q000138:chemical synthesis; D008745:Methylation; D010453:Peptide Synthases / Q000737:chemistry*; D000072417:Protein Domains; D015202:Protein Engineering / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D001688:Biological Products; D000249:Adenosine Monophosphate; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: domain swapping; enzyme catalysis; kutzneride; natural products; nonribosomal peptides; thiocoraline