An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A- and C-domains determines the product specificity of NRPSs.