Impact of Epimerization Domains on the Intermodular Transfer of Enzyme-Bound Intermediates in Nonribosomal Peptide Synthesis
| Autor: | Daniel B. Stein, Uwe Linne, Mohamed A. Marahiel, Martin Hahn |
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| Rok vydání: | 2006 |
| Předmět: |
Spectrometry
Mass Electrospray Ionization Stereochemistry Proteolysis Biochemistry Fourier transform ion cyclotron resonance chemistry.chemical_compound Nonribosomal peptide medicine Peptide bond Molecule Peptide Synthases Binding site Bifunctional Molecular Biology Tyrocidine chemistry.chemical_classification Binding Sites Molecular Structure medicine.diagnostic_test Organic Chemistry Peptide Fragments Recombinant Proteins Kinetics Enzyme chemistry Molecular Medicine Ribosomes |
| Zdroj: | ChemBioChem. 7:1807-1814 |
| ISSN: | 1439-4227 |
| DOI: | 10.1002/cbic.200600192 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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