title: Dipeptide formation on engineered hybrid peptide synthetases.
abstract: BACKGROUND
  Nonribosomal peptide synthetases (NRPSs) are modular 'megaenzymes' that catalyze the assembly of a large number of bioactive peptides using the multiple carrier thiotemplate mechanism. The modules comprise specific domains that act as distinct units to catalyze specific reactions associated with substrate activation, modification and condensation. Such an arrangement of biosynthetic templates has evoked interest in engineering novel NRPSs.
  
  
  RESULTS
  We describe the design and construction of a set of dimodular hybrid NRPSs. By introducing domain fusions between adenylation and thiolation (PCP) domains we designed synthetic templates for dipeptide formation. The predicted dipeptides, as defined by the specificity and arrangement of the adenylation domains of the constructed templates, were synthesized in vitro. The effect of the intramolecular fusion was investigated by determining kinetic parameters for substrate adenylation and thiolation. The rate of dipeptide formation on the artificial NRPSs is similar to that of natural templates.
  
  
  CONCLUSIONS
  Several new aspects concerning the tolerance of NRPSs to domain swaps can be deduced. By choosing the fusion site in the border region of adenylation and PCP domains we showed that the PCP domain exhibits no general substrate selectivity. There was no suggestion that selectivity of the condensation reaction was biased towards the donor amino acid, whereas at the acceptor position there was a size-determined selection. In addition, we demonstrated that a native elongation module can be converted to an initiation module for peptide-bond formation. These results represent the first example of rational de novo synthesis of small peptides on engineered NRPSs.
authors.affiliation: Philipps-Universit]at, Fachbereich Chemie/Biochemie, Marburg, 35032, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Doekel
authors.forename: M A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000096:biosynthesis*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004151:Dipeptides; D011993:Recombinant Fusion Proteins; D010453:Peptide Synthases