In Vivo Characterization of Tandem C‐Terminal Thioesterase Domains in Arthrofactin Synthetase

Macrocyclization of a peptide or a lipopeptide occurs at the last step of synthesis and is usually catalyzed by a single C-terminal thioesterase (Te) domain. Arthrofactin synthetase (Arf) from Pseudomonas sp. MIS38 represents a novel type of nonribosomal peptide synthetase that contains unique tandem C-terminal Te domains, ArfC_Te1 and ArfC_Te2. In order to analyze their function in vivo, site-directed mutagenesis was introduced at the putative active-site residues in ArfC_Te1 and ArfC_Te2. It was found that both Te domains were functional. Peaks corresponding to arthrofactin and its derivatives were absent in ArfC_Te1:S89A, ArfC_Te1:S89T, and ArfC_Te1:E26G/F27A mutants, and the production of arthrofactin by ArfC_Te2:S92A, ArfC_Te2:S92A/D118A, and ArfCDeltaTe2 was reduced by 95 % without an alteration of the cyclic lipoundecapeptide structure. These results suggest that Ser89 in ArfC_Te1 is essential for the completion of macrocyclization and the release of product. Glu26 and Phe27 residues are also part of the active site of ArfC_Te1. ArfC_Te2 might have been added during the evolution of Arf in order to improve macrocyclization efficiency.