# Defining structure and function of GT-A fold enzymes in bacterial glycan assembly

> **NIH NIH F32** · BOSTON UNIVERSITY (CHARLES RIVER CAMPUS) · 2024 · $74,284

## Abstract

Antibiotic resistance is a growing world problem and thus there is an urgent need to develop alternative means
to disable such pathogens. One target is the biosynthetic pathway of bacterial glycoconjugates, a diverse class
of macromolecules that play pivotal roles in cell-wall stability in challenging environments and in mediating
bacterial pathogen-host interactions. Structural information about the enzymes involved in the en bloc
construction of the glycoconjugates is lacking, which limits mechanism-based inhibitor design. This proposal
focuses on determining the structure-function relationships of the N-linked glycosylation pathway, because of
the high conservation of the pathway amongst the different pathogenic Campylobacter bacterium. The
glycosyltransferase PglI catalyzes the final step in glycan synthesis through attachment of a branching glycan to
the undecaprenyl phosphate-linked glycopolymer substrate. The branching position of the sugar varies widely
amongst the different species of Campylobacter and the mode of action of PglI is not known. PglI has an
annotated N-terminal GT-A fold domain and a C-terminal domain of unknown function. The additional domain
may play a role in controlling the location of the branching glycan by shaping the active site for acceptor sugar
binding and divalent cation binding and by mediating protein-protein or membrane-associate interactions. Aim 1
will identify the structural basis of selective glycan transfer in PglI enzymes through structural characterization of
PglI from several different Campylobacter species. Aim 2 will focus on determining the catalytic mechanism of
selective glycan transfer of PglI enzymes by kinetic characterization and mutagenesis studies. Aim 3 will focus
on the discovery of novel multidomain glycan biosynthetic enzymes through structural and functional profiling of
the GT-A fold superfamily. This aim will explore the structural space of GT-A fold enzymes by combining
bioinformatics with substrate screening and structural characterization. The structural characterization of PglI
will lead to an understanding of the mechanistic basis for branching glycan attachment in different Campylobacter
species, enabling structure-based design of inhibitors and ultimately new antibiotics. The results of this work will
advance the understanding of the molecular mechanisms that organize the multiprotein complexes of bacterial
glycoconjugate biosynthesis and allow for novel approaches for identifying chemical agents that disrupt these
pathogens.

## Key facts

- **NIH application ID:** 10904677
- **Project number:** 5F32GM149160-02
- **Recipient organization:** BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
- **Principal Investigator:** Hayley Knox
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $74,284
- **Award type:** 5
- **Project period:** 2023-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10904677

## Citation

> US National Institutes of Health, RePORTER application 10904677, Defining structure and function of GT-A fold enzymes in bacterial glycan assembly (5F32GM149160-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10904677. Licensed CC0.

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