Biosynthesis of crocacin involves an unusual hydrolytic release domain showing similarity to condensation domains.

The crocacins are potent antifungal and cytotoxic natural compounds from myxobacteria of the genus Chondromyces. Although total synthesis approaches have been reported, the molecular and biochemical basis guiding the formation of the linear crocacin scaffold has remained unknown. Along with the identification and functional analysis of the crocacin biosynthetic gene cluster from Chondromyces crocatus Cm c5, we here present the identification and biochemical characterization of an unusual chain termination domain homologous to condensation domains responsible for hydrolytic release of the product from the assembly line. In particular, gene inactivation studies and in vitro experiments using the heterologously produced domain CroK-C2 confirm this surprising role giving rise to the linear carboxylic acid. Additionally, we determined the kinetic parameters of CroK-C2 by monitoring hydrolytic cleavage of the substrate mimic N-acetylcysteaminyl-crocacin B using an innovative high-performance liquid chromatography mass spectrometry-based assay