title: Rational design of a bimodular model system for the investigation of heterocyclization in nonribosomal peptide biosynthesis.
abstract: Cyclization (Cy) domains in NRPS catalyze the heterocyclization of cysteine and serine/threonine to thiazoline and oxazoline rings. A model system consisting of the first two modules of bacitracin synthetase A fused to the thioesterase (Te) domain of tyrocidine synthetase was constructed (BacA1-2-Te) and shown to be active in production of the heterocyclic IleCys(thiazoline). Based on this model system, the feasibility of Cy domain module fusions was investigated by replacing the BacA2 Cy-A-PCP-module with modules of MbtB and MtaD from the biosynthesis systems of mycobactin and myxothiazol, revealing the formation of novel heterocyclic dipeptides. To dissect the reaction sequence of the Cy domain in peptide bond formation and heterocyclization, several residues of the BacA1-2-Te Cy domain were analyzed by mutagenesis. Two mutants exhibited formation of the noncyclic dipeptide, providing clear evidence for the independence of condensation and cyclization.
authors.affiliation: Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse, 35043 Marburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Duerfahrt
authors.forename: Katrin
authors.initials: K
authors.lastname: Eppelmann
authors.forename: Rolf
authors.initials: R
authors.lastname: Müller
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000818:Animals; D001483:Base Sequence; D003500:Cyclization; D004274:DNA, Recombinant / Q000378:metabolism; D004151:Dipeptides / Q000096:biosynthesis*; D015195:Drug Design; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D010080:Oxazoles / Q000032:analysis; D010170:Palmitoyl-CoA Hydrolase / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering; D016415:Sequence Alignment
chemical_list: D004274:DNA, Recombinant; D004151:Dipeptides; D009097:Multienzyme Complexes; D010080:Oxazoles; D010170:Palmitoyl-CoA Hydrolase; D010453:Peptide Synthases; C021774:bacitracin synthetase A; C104385:non-ribosomal peptide synthase; C035180:tyrocidine synthetase