Studies toward Understanding the Biosynthesis of Sactipeptides and the Creation of Peptide Natural Product Libraries through mRNA Display

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  • March 21, 2019
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  • Himes, Paul
    • Affiliation: Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry
Abstract
  • Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a class of natural products that are an attractive starting point for new antibiotics due to their wide range of structural diversity and biological activities. The post-translational modifications imparted upon the peptide substrate are carried out by promiscuous RiPP enzymes. Sactipeptides are members of the RiPPs family that are made through radical-mediated cysteine sulfur to α-carbon coupling reactions. The resulting thioether linkages give rise to sactipeptides defined structures and concomitant biological activities. The research presented here focuses on the biochemical and structural characterization of CteB, a radical SAM enzyme that imparts a single sactionine bridge, the development of an E. coli heterologous expression system for sactipeptides and the combination of RiPPs and mRNA display for the production of modified peptide libraries. We have biochemically and structurally characterized CteB, a radical SAM enzyme that imparts a sactionine bridge on its corresponding peptide substrate. A crystal structure was obtained at 2.04 Å and showed a RiPPs recognition element connected to a (β/α)6-TIM barrel fold, followed by an SPASM domain that houses two auxiliary [4Fe-4S] clusters, one of which contains a free coordination site for potential peptide ligation. We have developed an E. coli heterologous expression system for the production of sactipeptides based on subtilosin A from Bacillus subtilis 168. In the system, both the peptide substrate and radical SAM enzyme (AlbA) are expressed together and the modified sactipeptide is produced and isolated. This system was used to probe the substrate promiscuity of AlbA, and determine what changes it can tolerate. Additionally, an unnatural amino acid, O-Me-tyrosine, was able to be incorporated into the peptide substrate while also forming a thioether bridge at that position. We have also worked on combining the natural promiscuity of RiPPs enzymes with mRNA display to generate modified peptide libraries on a large scale (~5 x106). Using two previously described and characterized RiPPs systems, pantocin A and thiomuracin, we have used their respective RiPPs enzymes, PaaA and TbtF, to create RiPP peptide libraries to find elements important for binding and to further characterize the promiscuity of these modifying enzymes.
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  • In Copyright
Advisor
  • Frye, Stephen
  • Bowers, Albert
  • Kuhlman, Brian
  • Weeks, Kevin
  • Liu, Rihe
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2017
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