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.

Deciphering the Biosynthesis of Novel Class I Lanthipeptides from Marine Pseudoalteromonas Reveals a Dehydratase PsfB with Dethiolation Activity

“…31,32 Recently, Liu et al deciphered the biosynthetic elucidation of one representative linaridin member cypemycin, revealing that two membrane-associated enzymes, CypH and CypL, were synergistically responsible for 16 reactions, including 11 epimerizations, 4 dehydrations, and 1 dethiolation. 33 Neither of the dethiolation mechanisms mentioned above appeared related to the glutamyl-tRNA Glu -dependent way of PsfB shown in this study.…”

“…51 Recent studies by Liu et al identified that CypH and CypL are synergistically responsible for a series of 16 PTM reactions of cypemycin, including the dethiolation of Cys19, and exhibited high tolerance toward residues altering from Thr to Cys. 33 This enzymatic promiscuity may be attributed to the similar chemical properties of the sulfur and oxygen atoms. Dehydrated amino acids are among the most common unnatural amino acids in RiPPs and nonribosomal peptide synthetases.…”

“…The dethiolation of Cys has rarely been reported and only observed in linaridin biosynthesis and spirochaete flagella hook protein FlgE maturation . The dethiolation of Cys178 of FlgE was achieved by self-catalysis via β-elimination to produce Dha. , Recently, Liu et al deciphered the biosynthetic elucidation of one representative linaridin member cypemycin, revealing that two membrane-associated enzymes, CypH and CypL, were synergistically responsible for 16 reactions, including 11 epimerizations, 4 dehydrations, and 1 dethiolation . Neither of the dethiolation mechanisms mentioned above appeared related to the glutamyl-tRNA Glu -dependent way of PsfB shown in this study.…”

Enzymatic Generation of Thioaldehyde Motifs by Flavin-Dependent Cysteine Decarboxylases for Peptide Bioconjugation and Macrocyclization

“…A series of truncated MicA peptides, from 24-mer to 6-mer peptides, were examined, and all efficiently cyclized (Figures S21–25). Flavin-dependent cysteine decarboxylases usually display substrate tolerance toward peptides of diverse sequences. ,,,, Therefore, two short peptides with C-terminal cysteines but no sequence similarity to MicA, Pep-1 and Pep-2 , were synthesized by solid-phase synthesis and subjected to standard conditions. Both peptides were efficiently modified and cyclized (Figures S26 and 27).…”

Genome Mining of Myxopeptins Reveals a Class of Lanthipeptide-Derived Linear Dehydroamino Acid-Containing Peptides from Myxococcus sp. MCy9171

“…It is noteworthy that the catalytic mechanisms of these post-translational modifications (PTMs) are also distinguished from the biosynthesis of another class of dehydroamino acid-containing RiPPs, linaridins . Recently, Liu et al deciphered the cypemycin biosynthetic pathway, revealing that two membrane-associated enzymes, CypH and CypL, are synergistically responsible for not only the formation of characteristic Thr-derived dehydrobutyrines (Dhb) but also dethiolation and epimerizations . These enzymes appeared to be unrelated to the lanthipeptide biosynthetic machinery.…”