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Radical SAM Enzyme‐Catalyzed Formation of Thioether Crosslinks in Ribosomal‐Produced Peptides: Formation of the RiPP Natural Product Thuricin CD by the Radical SAM Enzymes TrnC and TrnD
Author(s) -
Jarrett Joseph T,
Holjencin Charles E
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1084.2
Subject(s) - thioether , chemistry , peptide , amino acid , cyclic peptide , cysteine , deubiquitinating enzyme , stereochemistry , enzyme , biochemistry , ubiquitin , gene
Bacteriocins are ribosomally‐produced peptide (RiPP) natural products produced by bacteria as highly effective antimicrobial natural products. Many bacteriocins are thought to function by selective insertion of multimers into the cell membrane, generating unregulated ion pores that ultimately trigger cell lysis. Bacteriocins often contain stabilizing post‐translational modifications including N‐ and C‐terminal additions, epimerized D‐amino acids, chemically modified amino acids, and disulfide and thioether crosslinks. Thuricin CD is an α 2 β 2 tetramer of two 30 residue peptides, Trnα and Trnβ, that are produced by Bacillus thuringiensis and specifically target the bacterium Clostridium difficile . Each of the peptides contains three thioether crosslinks in which cysteine residues are chemically bonded to the α‐carbon of distal amino acid residues; the resulting compact structure is resistant to proteolysis. The thioether crosslinks are proposed to be introduced by the Radical SAM enzymes TrnC and/or TrnD. In this work, we develop a fusion protein strategy for producing the propeptides TrnA and TrnB. We further demonstrate that TrnC alone is capable of introducing two of the thioether crosslinks. TrnC is shown to contain only one [4Fe‐4S] 2+ cluster, the radical SAM cluster, thus ruling out previously proposed mechanisms for radical thioether bond formation that require a second auxiliary FeS cluster. We propose a novel mechanism in which Radical SAM chemistry is used to activate the peptide backbone through formation of a peptide enolate radical prior to thioether bond formation. Two possible roles for TrnD are also discussed. The successful in vitro production of thuricin CD could provide a potential lead for novel antimicrobials targeting C. difficile infections.The radical SAM enzymes TrnC and TrnD are proposed to catalyze the formation of sulfur to α‐carbon crosslinks in the peptide natural product propeptides TrnA and TrnB.