Substrate plasticity of dehydratase SpaKC from the biosynthesis of thiosparsoamide.

ABSTRACT

Thioamitides are a group of ribosomally synthesized and post-translationally modified peptides that possess diverse bioactivities and are usually featured by thioamide and 2-aminovinyl-cysteine (AviCys) motifs. In natural product thiosparsoamide, the AviCys motif is formed by an enzyme cascade formed by the flavin-dependent decarboxylase SpaD and dehydratase SpaKC. SpaKC is a lanthipeptide synthetase homolog located outside the thiosparsoamide biosynthetic gene cluster. In this study, we show that SpaKC does not strictly require the N-terminal leader peptide of precursor peptide SpaA for substrate recognition and dehydration. The C-terminal seven residues serve as a minimal structural element for enzyme recognition. Through a systematic mutagenesis experiments, our study demonstrates the relaxed substrate specificity of SpaKC as a dehydratase and potentially as an enzymatic tool to install dehydroalanine or dehydrobutyrine motifs in peptides.