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

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.