Structural Elucidation of Thuricin CD, Thurincin H and a Leucocin A Mutant

  • Author / Creator
    Sit, Clarissa Sau-Wei
  • Thuricin CD, a two-component bacteriocin produced by Bacillus thuringiensis DPC 6431, exhibits potent activity against the hospital superbug Clostridium difficile ribotype O27. The two peptides (Trn-α and Trn-β) that constitute thuricin CD operate synergistically to kill sensitive bacteria at nanomolar concentrations. Characterization of Trn-α and Trn-β by mass spectrometry established that each peptide is 6 mass units lighter than predicted from the sequence of its structural gene, suggesting that the peptides undergo post-translational modification. Analysis of nuclear Overhauser effect (NOE) data from NMR experiments run on [13C, 15N]Trn-α and [13C, 15N]Trn-β indicate that each peptide features three sulfur to α-carbon (S-Cα) thioether bridges between Cys5 and residue 28, Cys9 and residue 25, and Cys13 and residue 21. To elucidate the stereochemistry of these bridges, the 3D structures of the eight possible stereoisomers for each peptide were calculated and compared to determine which stereoisomer best fit the NOE data. The most representative structures of Trn-α and Trn-β both feature L-stereochemistry at residue 21 (α-R), L-stereochemistry at residue 25 (α-R), and D-stereochemistry at residue 28 (α-S). Thurincin H is a single-component bacteriocin produced by B. thuringiensis SF361 that is highly active against the human pathogen Listeria monocytogenes. Mass spectrometry analysis revealed that thurincin H is 8 mass units lighter than expected from its genetic sequence. NOE experiments on [13C, 15N]thurincin H provided evidence for the presence of four S-Cα bridges in the peptide. Out of the 16 possible stereoisomers, the stereoisomer that features D-stereochemistry (α-S) at all four sulfur-linked α-carbons gave the most representative 3D solution structure of thurincin H. Leucocin A is an antilisterial bacteriocin with a disulfide bridge between Cys9 and Cys14. Previous studies showed that replacement of the cysteines with phenylalanines had no effect on the peptide’s activity, while replacement with serines resulted in complete loss of activity. [13C, 15N]-(C9S, C14S)-leucocin A, produced by heterologous expression, gave an elongated C-terminal α-helix and a disordered N-terminus compared to the 3D structure of wild-type leucocin A, thus providing a potential explanation for the loss in activity. Studies are underway to determine the 3D structure of (C9F, C14F)-leucocin A.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Vederas, John C. (Chemistry)
  • Examining committee members and their departments
    • Loppnow, Glen R. (Chemistry)
    • Begley, Tadhg P. (Chemistry)
    • McMullen, Lynn M. (Agricultural, Food and Nutritional Sciences)
    • West, Frederick G. (Chemistry)
    • Vederas, John C. (Chemistry)
    • Campbell, Robert E. (Chemistry)