# Structure and function of the monotopic phosphoglycosyl transferase superfamily: Initiators of biosynthesis of complex bacterial glycoconjugates

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $318,411

## Abstract

Summary
Complex glycoconjugates play a pivotal role in bacterial survival, colonization and virulence and thus, to
the interactions between symbiotic and pathogenic bacteria and their human hosts. An important
mechanism for the assembly of complex structures involves initiation of glycan assembly on the
cytoplasmic face of cell membranes catalyzed by polyprenol phosphate (PrenP) phosphoglycosyl
transferases (PGTs). PGTs catalyze transfer of a C1’-phosphosugar moiety from a soluble nucleoside
diphosphate-activated donor to a PrenP acceptor, yielding a membrane-bound polyprenol
diphosphosugar. The proposed studies focus on a PGT superfamily with a monotopic membrane topology
for which, until our recent studies, there has been only limited structural and mechanistic information.
These enzymes differ in structure, mechanism and topology from the well-known polytopic PGTs.
Biochemical studies together with the recently-determined structure of Campylobacter concisus PglC from
our laboratories, show that the monotopic PGTs include a reentrant membrane helix (RMH) that penetrates
only one leaflet of the bilayer then re-emerges. Aim 1 will Identify sugar-specificity determinants for all
three families within the monotopic PGT superfamily by determining X-ray crystal structures of liganded
complexes with nucleoside diphosphate sugar substrates. This will enable assignment of specificity of
newly-identified monotopic PGTs and provide information on the function of PGTs in the glycoconjugate
biosynthetic pathways of various pathogens. In Aim 2 the model that binding of the UDP-sugar substrate
triggers the movement of the soluble residue loop (aa 61-80) to complete substrate-binding determinants
and close the active site for catalysis will be tested using cross-linking and fluorescence-based
approaches. The sequence of the RMH integral to membrane interaction will be used to develop HMMs to
identify similar RMH via bioinformatics within the monotopic PGT superfamily and then applied to unrelated
proteins predicted to have RMHs, those potentially misannotated as bitopic and other integral membrane
proteins. Aim 3 develops nucleoside derivatives that will serve as fluorescent probes, activity-based protein
profiling probes and inhibitors of the monotopc PGT superfamily. Overall the in-depth study of the substrate
specificities and functions of the monotopic superfamily and design of biological probes will establish the
fundamental knowledge and tools needed for validating and intervening in the action of potential
therapeutic targets.

## Key facts

- **NIH application ID:** 9853808
- **Project number:** 5R01GM131627-02
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Karen N. Allen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $318,411
- **Award type:** 5
- **Project period:** 2019-02-01 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9853808

## Citation

> US National Institutes of Health, RePORTER application 9853808, Structure and function of the monotopic phosphoglycosyl transferase superfamily: Initiators of biosynthesis of complex bacterial glycoconjugates (5R01GM131627-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9853808. Licensed CC0.

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