Reconstitution of the Linaridin Pathway Provides Access to the Family-Determining Activity of Two Membrane-Associated Proteins in the Formation of Structurally Underestimated Cypemycin.

Abstract

Cypemycin is a parent linaridin peptide known to contain nonproteinogenic dehydrobutyrine, N,N-dimethylalanine, and aminovinyl-cysteine residues. The enzymatic process by which this ribosomally synthesized peptide is formed remains elusive largely because of the deficiency of knowledge in post-translational modifications (PTMs) conducted by CypH and CypL, the two membrane-associated enzymes unique to linaridin biosynthesis. Based on heterologous reconstitution of the pathway in Streptomyces coelicolor, we report the detailed structural characterization of cypemycin as a previously unknown, d-amino acid-rich linaridin. In particular, the unprecedented family-determining activity of CypH and CypL was revealed, which, in addition to hydrolysis for removal of the N-terminal leader peptide, leads to transformation of the core peptide part of the precursor peptide through mechanistically related 16 reactions for residue epimerization (11 amino acids), dehydration (4 Thr), and dethiolation (Cys19). Subsequent functionalization for linaridin maturation includes CypD-involved aminovinyl-cysteine formation and N,N-dimethylation of the newly exposed N-terminal d-Ala residue that requires CypM activity. Genetic, chemical, biochemical, engineering, and modeling approaches were used to access the structure of cypemycin and the versatility of the CypH and CypL combination that is achieved in catalysis. This work furthers the appreciation of PTM chemistry and facilitates efforts for expanding linaridin structural diversity using synthetic biology methods.