Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes
Monotopic phosphoglycosyl transferase enzymes (MonoPGTs) are membrane-bound enzymes that initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, l...
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| Format: | Thesis |
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Massachusetts Institute of Technology
2023
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| Online Access: | https://hdl.handle.net/1721.1/151506 |
| _version_ | 1826189034554130432 |
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| author | Anderson, Alyssa J. |
| author2 | Imperiali, Barbara |
| author_facet | Imperiali, Barbara Anderson, Alyssa J. |
| author_sort | Anderson, Alyssa J. |
| collection | MIT |
| description | Monotopic phosphoglycosyl transferase enzymes (MonoPGTs) are membrane-bound enzymes that initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, limited biochemical characterization of this enzyme superfamily has been performed. To close the gap between sequence annotation and biochemical characterization of these critical enzymes, this thesis presents methods for characterizing the substrate specificities, interactions, and regulatory mechanisms of monoPGTs.
Substrate specificities of monoPGTs are largely unknown; therefore, connecting the sequences of monoPGTs to the composition of the final glycoconjugate produced remains a significant challenge. In Chapter 2, structural, sequence, and biochemical analyses are combined to identify co-conserved sequence “fingerprints” that predict the substrate specificity for a subset of monoPGTS that utilize UDP-N,N’-diacetylbacillosamine. The methodology described is generalizable and may be applied to identify sequence motifs that serve as fingerprints for monoPGTs of differing substrate specificities.
Evidence suggests glycoconjugate biosynthesis machinery forms complexes to centralize glycoconjugate production in the cell. However, the protein-protein interactions that comprise these complexes are poorly understood. In Chapter 3, I explore interactions among a subfamily of monoPGTs known as large (Lg) PGTs in near-native membrane mimetic systems. In this work, I characterize LgPGT dimerization using detergent-free solubilization and chemical crosslinking. This approach to studying protein-protein interactions of membrane-bound enzymes can be broadly applied to characterize interactions among glycoconjugate biosynthesis enzymes as a whole.
The regulatory mechanisms that govern monoPGT regulation remain enigmatic. In Chapter 4, activity-based protein profiling (ABPP) probes are implemented as protein-centric, membrane-protein compatible tools that lay the groundwork for understanding the activity and regulation of the monoPGT superfamily from a cellular proteome. Robust, covalent labeling at the active site of various representative monoPGTs from unfractionated cell membrane fractions is demonstrated using 3- phenyl-2H-azirine probes. |
| first_indexed | 2024-09-23T08:08:31Z |
| format | Thesis |
| id | mit-1721.1/151506 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T08:08:31Z |
| publishDate | 2023 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1515062023-08-01T04:23:15Z Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes Anderson, Alyssa J. Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Monotopic phosphoglycosyl transferase enzymes (MonoPGTs) are membrane-bound enzymes that initiate the assembly of prokaryotic glycoconjugates essential for bacterial survival and proliferation. MonoPGTs belong to an expansive superfamily with a diverse and richly annotated sequence space; however, limited biochemical characterization of this enzyme superfamily has been performed. To close the gap between sequence annotation and biochemical characterization of these critical enzymes, this thesis presents methods for characterizing the substrate specificities, interactions, and regulatory mechanisms of monoPGTs. Substrate specificities of monoPGTs are largely unknown; therefore, connecting the sequences of monoPGTs to the composition of the final glycoconjugate produced remains a significant challenge. In Chapter 2, structural, sequence, and biochemical analyses are combined to identify co-conserved sequence “fingerprints” that predict the substrate specificity for a subset of monoPGTS that utilize UDP-N,N’-diacetylbacillosamine. The methodology described is generalizable and may be applied to identify sequence motifs that serve as fingerprints for monoPGTs of differing substrate specificities. Evidence suggests glycoconjugate biosynthesis machinery forms complexes to centralize glycoconjugate production in the cell. However, the protein-protein interactions that comprise these complexes are poorly understood. In Chapter 3, I explore interactions among a subfamily of monoPGTs known as large (Lg) PGTs in near-native membrane mimetic systems. In this work, I characterize LgPGT dimerization using detergent-free solubilization and chemical crosslinking. This approach to studying protein-protein interactions of membrane-bound enzymes can be broadly applied to characterize interactions among glycoconjugate biosynthesis enzymes as a whole. The regulatory mechanisms that govern monoPGT regulation remain enigmatic. In Chapter 4, activity-based protein profiling (ABPP) probes are implemented as protein-centric, membrane-protein compatible tools that lay the groundwork for understanding the activity and regulation of the monoPGT superfamily from a cellular proteome. Robust, covalent labeling at the active site of various representative monoPGTs from unfractionated cell membrane fractions is demonstrated using 3- phenyl-2H-azirine probes. Ph.D. 2023-07-31T19:44:58Z 2023-07-31T19:44:58Z 2023-06 2023-07-13T14:05:14.622Z Thesis https://hdl.handle.net/1721.1/151506 In Copyright - Educational Use Permitted Copyright retained by author(s) https://rightsstatements.org/page/InC-EDU/1.0/ application/pdf Massachusetts Institute of Technology |
| spellingShingle | Anderson, Alyssa J. Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| title | Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| title_full | Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| title_fullStr | Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| title_full_unstemmed | Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| title_short | Insights into the substrate specificities, interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| title_sort | insights into the substrate specificities interactions and regulatory mechanisms of bacterial glycoconjugate biosynthesis enzymes |
| url | https://hdl.handle.net/1721.1/151506 |
| work_keys_str_mv | AT andersonalyssaj insightsintothesubstratespecificitiesinteractionsandregulatorymechanismsofbacterialglycoconjugatebiosynthesisenzymes |