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Dissertation
Characterization of adenylating enzymes and domain interactions involved in insect and bacterial non-ribosomal peptide synthesis
Doctor of Philosophy (Ph.D.), Drexel University
Aug 2021
DOI:
https://doi.org/10.17918/00000859
Abstract
Non-ribosomal peptide synthetases (NRPSs) are responsible for full or partial biosynthesis of medically invaluable compounds including antifungals, immunosuppressants, and antibiotics like vancomycin. Synthetases have a modular architecture and function in an assembly line manner where peptide products are extended and modified as they are passed between modules. Engineering synthetases by replacing or re-ordering modules is an attractive strategy to generate "unnatural natural products". However, successful combinatorial engineering requires characterization of new NRPS domains and an improved understanding of the protein-protein interactions that govern synthetase function. This work focused on determining domain composition and substrate specificity of three-domain synthetases from bacterial species Lactobacillus iners and Scytonema hofmannii. Sequence analysis predicted the bacterial synthetases to contain adenylation- and carrier protein-domains, as well as C-terminal domains with hypothetical functions. We adopted a recently published mass spectrometry method to probe substrate specificity of these A-domains and they were shown to activate proline and glycine. Sequence homology between bacterial three-domain NRPSs and the Drosophila melanogaster Ebony synthetase, led us to interrogate Ebony in more detail and we focused on protein-protein interactions between domains within the synthetase. Dissection of the synthetase into separate proteins showed that certain combinations failed to produce product, but nine percent of activity was preserved following dissection of Ebony into a di-domain with detached C-terminal domain. Collectively, this work contributes to NRPS engineering efforts by characterizing the specificity of new domains and developing a platform to assess activity of a dissected synthetase. This information can be leveraged to utilize three-domain synthetases as engineering tools to characterize additional domains and generate new dipeptides.
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Details
- Title
- Characterization of adenylating enzymes and domain interactions involved in insect and bacterial non-ribosomal peptide synthesis
- Creators
- Amanda Jo Platt
- Contributors
- Joris Beld (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xv, 155 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991015473792404721