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Permanent link (DOI): https://doi.org/10.7939/R3BM39
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Structural Elucidation of Thuricin CD, Thurincin H and a Leucocin A Mutant Open Access
- Other title
nuclear magnetic resonance
- Type of item
- Degree grantor
University of Alberta
- Author or creator
Sit, Clarissa Sau-Wei
- Supervisor and department
Vederas, John C. (Chemistry)
- Examining committee member and department
Begley, Tadhg P. (Chemistry)
Vederas, John C. (Chemistry)
McMullen, Lynn M. (Agricultural, Food and Nutritional Sciences)
Loppnow, Glen R. (Chemistry)
Campbell, Robert E. (Chemistry)
West, Frederick G. (Chemistry)
Department of Chemistry
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
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.
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- Citation for previous publication
Rea, M. C.; Sit, C. S.; Clayton, E.; O'Connor, P. M.; Whittal, R. M.; Zheng, J.; Vederas, J. C.; Ross, R. P.; Hill, C. Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile. Proc. Natl. Acad. Sci. U. S. A., 2010, 107 (20), 9352-9357.Sit, C. S.; van Belkum, M. J.; McKay, R. T.; Worobo, R. W.; Vederas, J. C. The 3D solution structure of thurincin H, a bacteriocin with four sulfur to alpha-carbon crosslinks. Angew. Chem.-Int. Ed., 2011, Accepted.Sit, C. S.; McKay, R. T.; Hill, C.; Ross, R. P.; Vederas, J. C. The 3D structure of thuricin CD, a two-component bacteriocin with cysteine sulfur to alpha-carbon crosslinks. J. Am. Chem. Soc., 2011, 133 (20), 7680-7683.
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