Hydroxy Acid Activation in Fungal Non‐Ribosomal Peptide Synthesis Assessed by Site‐Directed Mutagenesis

Abstract The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N ‐methylated l ‐amino and d ‐hydroxy acids. They are synthesized by non‐ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A 1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non‐amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.