# Structural Characterization of AdoMet Radical Enzyme-Catalyzed Posttranslational Modifications in Bacterial Anaerobic Metabolism

> **NIH NIH F32** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2021 · $56,747

## Abstract

Project Summary
Enzymes that install posttranslational modifications (PTMs) on bacterial peptides and proteins are integral in
cellular functions such as the formation of bioactive peptide natural products and the activation of enzymes
important for bacterial adaptation to oxygen-limited environments. Understanding the process of PTM
formation can inform on the engineering of novel peptide therapeutics and on the methods of bacterial
colonization of host environments in infection. S-adenosyl-L-methionine (AdoMet) radical enzymes produce
numerous PTMs that change the functionality of the targeted residue(s). AdoMet radical enzymes perform
oxygen-sensitive, site-selective radical chemistry on macromolecular substrates, yet a structural understanding
of how they accomplish this impressive chemistry has lagged behind in the analysis of the AdoMet radical
enzyme superfamily, with no complete AdoMet radical enzyme-protein complex fully visualized. The aims of
this proposal include structural characterization of two AdoMet radical enzymes that modify the Cα of specific
amino acids within their large substrates: 1) an AdoMet radical epimerase with a peptide substrate and 2)
pyruvate formate lyase activase (PFL-AE) in complex with its partner PFL. The epimerase irreversibly converts
L-amino acids to D-amino acids within a ribosomal peptide, thus altering the final conformation and influencing
its bioactivity. Determining how one epimerase positions substrate to perform multiple turnovers at specified
residues will require structural insight. X-ray crystallography will be used to examine interactions of AdoMet
radical epimerases with peptide substrates. PFL-AE forms the catalytically essential glycyl radical on PFL to
make formate and acetyl-CoA from pyruvate and CoA. How PFL-AE contacts PFL and how the glycyl radical
transitions from the PFL-AE active site to the buried PFL active site remain to be elucidated. X-ray
crystallography and electron microscopy will be used to determine structures of PFL-AE in complex with PFL.
Structural analysis of both systems will provide much needed insight into interactions required for construction
of a protein complex that performs site-selective oxygen-sensitive radical-generating chemistry.

## Key facts

- **NIH application ID:** 10246524
- **Project number:** 5F32GM133056-03
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Emily Ulrich
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $56,747
- **Award type:** 5
- **Project period:** 2019-09-01 → 2022-06-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10246524

## Citation

> US National Institutes of Health, RePORTER application 10246524, Structural Characterization of AdoMet Radical Enzyme-Catalyzed Posttranslational Modifications in Bacterial Anaerobic Metabolism (5F32GM133056-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10246524. Licensed CC0.

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