Exploring the impact of different thioesterase domains for the design of hybrid peptide synthetases

Background: A large number of pharmacologically important peptides are synt hesized by multifunctional enzymes, the nonribosomal peptide synthetases (N RPSs). The thioesterase (Te) domain tit the C-terminus of the last NRPS cat alyzes product cleavage by hydrolysis or complex macrocyclization. Recent s tudies with excised Te domains and peptidyl-S-N-acetyl cysteamine substrate substitutes led to substantial insights in terms of cyclization activity a nd substrate tolerance of these enzymes. Their use in engineered hybrid NRP Ss is an interesting but yet only little explored target for approaches to achieve new structural diversity and designed products. Results: To study the capability of various Te domains to function in hybri d NRPSs, six different Te domains that catalyze different modes of terminat ion in their natural systems were fused to a bimodular model NRPS system, c onsisting of the first two modules of tyrocidine NRPS, TycA and ProCAT. All Te domains were active in hydrolyzing the enzymatically generated dipeptid e substrate D-Phe-Abu from the NRPS template with, however, greatly varying turnover rates. Two Te domains were also capable of hydrolyzing the substr ate D-Phe-Pro and partially cyclized the D-Phe-Abu dipeptide, indicating th at in an artificial context Te domains may display hydrolytic and cyclizati on activities that are not easily predictable. Conclusions: Te domains from heterologous NRPSs can be utilized for the con struction of hybrid NRPSs. This is the first comparative study to explore t heir influence on the product pattern. The inherent specificity and regiose lectivity of Te domains should allow control of the desired product cleavag e, but can also lead to other modes of termination potentially useful for g enerating structural diversity. Our results provide the first data for choo sing the proper Te domain for a particular termination reaction. (C) 2001 E lsevier Science B.V. All rights reserved.