Biochemical Characterization of Glycan Assembly Pathway Enzymes
Glycans and glycoconjugates are found in all domains of life and are known to mediate many important and diverse biological processes. Glycan biosynthesis is not templated, and complex glycans are instead assembled en bloc in highly specific and tightly regulated enzymecatalyzed assembly pathways th...
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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/1721.1/151626 https://orcid.org/0000-0003-0871-0376 |
| _version_ | 1826212092217131008 |
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| author | Bernstein, Hannah |
| author2 | Imperiali, Barbara |
| author_facet | Imperiali, Barbara Bernstein, Hannah |
| author_sort | Bernstein, Hannah |
| collection | MIT |
| description | Glycans and glycoconjugates are found in all domains of life and are known to mediate many important and diverse biological processes. Glycan biosynthesis is not templated, and complex glycans are instead assembled en bloc in highly specific and tightly regulated enzymecatalyzed assembly pathways that occur at the membrane interface. Despite the critical roles of glycans, our understanding of the glycome is quite incomplete. This thesis describes a biochemical approach that may be used to provide greater insight into both glycan structure elucidation as well as the structural determinants of substrate specificity for these glycanbiosynthesizing enzymes.
Phosphoglycosyl transferases (PGTs) initiate glycan assembly pathways by catalyzing the transfer of a phospho-sugar from a nucleotide-diphosphate (NDP)-sugar to a polyprenol phosphate (PrenP), yielding a Pren-PP-sugar product that is then elaborated by sequential glycosyl transferases (GTs), flipped across the membrane, and transferred to the final target. Previous work has defined two PGT superfamilies, which are defined by different membrane topologies and catalytic mechanisms, and characterized the requirements of the PrenP substrate, but less is known about the structural determinants of substrate specificity with respect to the NDP-sugar substrate. This thesis presents the methods used to synthesize and purify a panel of candidate UDP-sugar substrates that can be deployed in various binding or activity studies in order to investigate the structure-activity relationship of PGT enzymes and NDP-sugar substrates in vitro. This library is then used to biochemically validate the reported substrate specificity profile of several monotopic PGTs.
The next section of this thesis focuses on GTs enzymes, which are also known to demonstrate incredible selectivity with respect to the NDP-sugar substrate. Because GTs adopt one of only a few different folds, elucidation of the structural determinants of substrate specificity could have wide-reaching impacts. The substrate specificity of GTs in the Pgl pathway of two Campylobacter concisus strains is probed as a model system. Finally, the utility of this biochemical approach is exemplified through its application in identifying the first two monosaccharides of the exopolysaccharide (EPS) glycan from Bacillus subtilis, which had not been previously known. |
| first_indexed | 2024-09-23T15:16:16Z |
| format | Thesis |
| id | mit-1721.1/151626 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T15:16:16Z |
| publishDate | 2023 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/1516262023-08-01T03:12:50Z Biochemical Characterization of Glycan Assembly Pathway Enzymes Bernstein, Hannah Imperiali, Barbara Massachusetts Institute of Technology. Department of Biology Glycans and glycoconjugates are found in all domains of life and are known to mediate many important and diverse biological processes. Glycan biosynthesis is not templated, and complex glycans are instead assembled en bloc in highly specific and tightly regulated enzymecatalyzed assembly pathways that occur at the membrane interface. Despite the critical roles of glycans, our understanding of the glycome is quite incomplete. This thesis describes a biochemical approach that may be used to provide greater insight into both glycan structure elucidation as well as the structural determinants of substrate specificity for these glycanbiosynthesizing enzymes. Phosphoglycosyl transferases (PGTs) initiate glycan assembly pathways by catalyzing the transfer of a phospho-sugar from a nucleotide-diphosphate (NDP)-sugar to a polyprenol phosphate (PrenP), yielding a Pren-PP-sugar product that is then elaborated by sequential glycosyl transferases (GTs), flipped across the membrane, and transferred to the final target. Previous work has defined two PGT superfamilies, which are defined by different membrane topologies and catalytic mechanisms, and characterized the requirements of the PrenP substrate, but less is known about the structural determinants of substrate specificity with respect to the NDP-sugar substrate. This thesis presents the methods used to synthesize and purify a panel of candidate UDP-sugar substrates that can be deployed in various binding or activity studies in order to investigate the structure-activity relationship of PGT enzymes and NDP-sugar substrates in vitro. This library is then used to biochemically validate the reported substrate specificity profile of several monotopic PGTs. The next section of this thesis focuses on GTs enzymes, which are also known to demonstrate incredible selectivity with respect to the NDP-sugar substrate. Because GTs adopt one of only a few different folds, elucidation of the structural determinants of substrate specificity could have wide-reaching impacts. The substrate specificity of GTs in the Pgl pathway of two Campylobacter concisus strains is probed as a model system. Finally, the utility of this biochemical approach is exemplified through its application in identifying the first two monosaccharides of the exopolysaccharide (EPS) glycan from Bacillus subtilis, which had not been previously known. Ph.D. 2023-07-31T19:54:05Z 2023-07-31T19:54:05Z 2023-06 2023-07-13T14:05:28.472Z Thesis https://hdl.handle.net/1721.1/151626 https://orcid.org/0000-0003-0871-0376 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 | Bernstein, Hannah Biochemical Characterization of Glycan Assembly Pathway Enzymes |
| title | Biochemical Characterization of Glycan Assembly Pathway Enzymes |
| title_full | Biochemical Characterization of Glycan Assembly Pathway Enzymes |
| title_fullStr | Biochemical Characterization of Glycan Assembly Pathway Enzymes |
| title_full_unstemmed | Biochemical Characterization of Glycan Assembly Pathway Enzymes |
| title_short | Biochemical Characterization of Glycan Assembly Pathway Enzymes |
| title_sort | biochemical characterization of glycan assembly pathway enzymes |
| url | https://hdl.handle.net/1721.1/151626 https://orcid.org/0000-0003-0871-0376 |
| work_keys_str_mv | AT bernsteinhannah biochemicalcharacterizationofglycanassemblypathwayenzymes |