Dipeptide formation on engineered hybrid peptide synthetases

Background: Nonribosomal peptide synthetases (NRPSs) are modular 'megaenzym es' that catalyze the assembly of a large number of bioactive peptides usin g the multiple carrier thiotemplate mechanism. The modules comprise specifi c domains that act as distinct units to catalyze specific reactions associa ted with substrate activation, modification and condensation. Such an arran gement of biosynthetic templates has evoked interest in engineering novel N RPSs. Results: We describe the design and construction of a set of dimodular hybr id 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 k inetic parameters for substrate adenylation and thiolation. The rate of dip eptide formation on the artificial NRPSs is similar to that of natural temp lates. Conclusions: Several new aspects concerning the tolerance of NRPSs to domai n 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 gene ral substrate selectivity. There was no suggestion that selectivity of the condensation reaction was biased towards the donor amino acid, whereas at t he acceptor position there was a size-determined selection. In addition, we demonstrated that a native elongation module can be converted to an initia tion module for peptide-bond formation. These results represent the first e xample of rational de novo synthesis of small peptides on engineered NRPSs.