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Proteome-Wide Bioinformatic Annotation and Functional Validation of the Monotopic Phosphoglycosyl Transferase Superfamily
Open accessPreprint — published as 10.1073/pnas.2417572121
Abstract
Phosphoglycosyl transferases (PGTs) are membrane proteins that initiate glycoconjugate biosynthesis by transferring a phospho-sugar moiety from a soluble nucleoside diphosphate sugar to a membrane-embedded polyprenol phosphate acceptor. The centrality of PGTs in complex glycan assembly and the current lack of functional information make these enzymes high-value targets for biochemical investigation. In particular, the small monotopic PGT family is exclusively bacterial and represents the minimal functional unit of the monotopic PGT superfamily. Here, we combine a sequence similarity network (SSN) analysis with a generalizable, luminescence-based activity assay to probe the substrate specificity of this family of monoPGTs in a bacterial cell-membrane fraction. This strategy allows us to identify specificity on a far more significant scale than previously achievable and correlate preferred substrate specificities with predicted structural differences within the conserved monoPGT fold. Finally, we present the proof-of-concept for a small-scale inhibitor screen (eight nucleoside analogs) with four monoPGTs of diverse substrate specificity, thus building a foundation for future inhibitor discovery initiatives. Significance: Uncovering the function and specificity of enzymes responsible for glycoconjugate biosynthesis traditionally requires a multi-faceted and individually curated approach. This is especially true for bacterial glycoconjugates due to greater monosaccharide diversity and a paucity of established structural information. Here we leverage bioinformatic and in-vitro tools to predict and validate substrate specificity for a unique, exclusively bacterial family of enzymes responsible for the first step in many of these glycan assembly pathways. We further show that this platform is suitable for enhanced functional annotation and inhibitor testing, paving the way for the development of urgently needed antibiotics.
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Metadata
- Authors
- Durand, T., Dodge, G. J., Siuda, R. P., Higinbotham, H. R., Arbour, C. A., Ghosh, S., Allen, K. N., & Imperiali, B.
- Affiliations
- Imperial College London (GB)
- Biogen (United States) (US)
- Boston University (US)
- Massachusetts Institute of Technology (US)
- Corresponding author
- Allen, Karen N.
- Imperiali, Barbara
- ORCID
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- openRxiv
- Published
- 2024-07-11
- Volume
- 121
- Issue
- 49
- Pages
- e2417572121-e2417572121
- ISSN
- 0027-8424 (ISSN-L)
- PMC
- PMC11626204
- OpenAlex
- W4404791536
- Cited by
- 6
- References
- 1
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 1
- FWCI
- 0.81
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- Funders
- National Institutes of Health
- National Institute of General Medical Sciences (F32GM134576, F32GM136023, R01GM131627)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
- Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Keywords
- substrate specificity, sequence similarity networks, nucleoside diphosphate sugars, inhibitor screening, nucleoside analogues, protein structure-function relationship
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