title: Impact of epimerization domains on the intermodular transfer of enzyme-bound intermediates in nonribosomal peptide synthesis.
abstract: Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.
authors.affiliation: Fachbereich Chemie/Biochemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Stein
authors.forename: Uwe
authors.initials: U
authors.lastname: Linne
authors.forename: Martin
authors.initials: M
authors.lastname: Hahn
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D001665:Binding Sites; D007700:Kinetics; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014440:Tyrocidine / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D014440:Tyrocidine; D010453:Peptide Synthases; C035180:tyrocidine synthetase