Insights into pyrazinone biosynthesis from biochemical and cryo-EM studies of the hybrid NRPS-PKS enzyme CorA

Hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) systems integrate peptide and polyketide biosynthetic logic to assemble complex natural products. CorA, a prototypical NRPS-PKS hybrid from the myxobacterium Corallococcus exiguus SDU70, is responsible for producing 5-alkylated pyrazinones, a family of bioactive metabolites with morphogenetic roles in myxobacterial development. Previous studies have identified CorA as a core biosynthetic enzyme responsible for the formation of 5-methylated pyrazinones; however, the structural and mechanistic basis underlying its capacity to recognize diverse dipeptide substrates and accommodate distinct acyl-CoA extender units remains elusive. Here, combining in vitro biochemistry, structure determination by cryo-electron microscopy (EM), mutagenesis, and molecular docking, we show that full-length CorA generates twelve distinct 5-alkylated pyrazinone products using six amino acid combinations and two acyl-CoA extenders, highlighting an unusually permissive gating mechanism within the KS domain. The 3.1-Å cryo-EM structure of the CorA KS-AT didomain provides structural insights into its substrate-binding architecture. The atypical SGFH motif of the AT domain permits dual binding of M-CoA and MM-CoA, and an irregular non-helical loop in the KS domain creates an open pocket for large amino acid-derived intermediates. These distinctive architectural features confer robust substrate promiscuity on CorA. Collectively, our structural data offers insights to understand the wide substrate compatibility of NRPS-PKS hybrid enzymes and may offer useful clues for the rational engineering of biosynthetic pathways to diversify natural product scaffolds.