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The radical SAM enzyme EpeE exhibits distinct site reactivity during the biosynthesis of the RiPP natural product epipeptide.

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Title: The radical SAM enzyme EpeE exhibits distinct site reactivity during the biosynthesis of the RiPP natural product epipeptide.
Authors: Walls, William G.1, Yang, Hao2, Ho, Madeline B.2, Broderick, William E.1, Hoffman, Brian M.2, Broderick, Joan B.1 jbroderick@montana.edu
Source: Proceedings of the National Academy of Sciences of the United States of America. 3/24/2026, Vol. 123 Issue 12, p1-11. 61p.
Subjects: Epimerization, Electron paramagnetic resonance, Adenosylmethionine, Peptide synthesis, Ligand binding (Biochemistry), Epimerases, Natural products
Abstract: Radical S-adenosyl-l-methionine (SAM) enzymes figure prominently in the formation of ribosomally synthesized and posttranslationally modified peptides (RiPPs), where they catalyze peptide modifications including epimerization, thioether crosslink formation, and peptide backbone splicing. Here, we use rapid freeze-quench trapping together with electron paramagnetic resonance and electron-nuclear double resonance techniques to probe the mechanistic steps of the two epimerization reactions catalyzed by the radical SAM enzyme EpeE during conversion of its peptide substrate to the epipeptide natural product. Use of the EpeE C223S variant facilitated trapping and characterization of Ca radical intermediates, supporting a central role for C223 in the proposed epimerization mechanism. We showed that both wild-type and C223S EpeE with bound SAM and peptide substrate form the organometallic intermediate O upon reaction, and that thermal annealing of O results in conversion to an organic radical intermediate. Freeze-quenching at longer times allowed us to directly trap the organic radical intermediate, and isotopic labeling together with use of substrate variants allowed for detailed characterization of the substrate radical intermediates. The results revealed that while LC-MS enzymatic assays point to Ile12 as the initial site of epimerization, freeze-quench EPR reveals that Val4 is the preferred site for initial Ca radical formation. These apparently conflicting results were resolved by the observation that the Ile12 Ca radical is more efficiently quenched to form the d-Ile, thus providing insights into the determinants for substrate binding and epimerization by EpeE. [ABSTRACT FROM AUTHOR]
Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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PubType: Academic Journal
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  Data: The radical SAM enzyme EpeE exhibits distinct site reactivity during the biosynthesis of the RiPP natural product epipeptide.
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  Data: <searchLink fieldCode="AR" term="%22Walls%2C+William+G%2E%22">Walls, William G.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Yang%2C+Hao%22">Yang, Hao</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Ho%2C+Madeline+B%2E%22">Ho, Madeline B.</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Broderick%2C+William+E%2E%22">Broderick, William E.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Hoffman%2C+Brian+M%2E%22">Hoffman, Brian M.</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Broderick%2C+Joan+B%2E%22">Broderick, Joan B.</searchLink><relatesTo>1</relatesTo><i> jbroderick@montana.edu</i>
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  Data: <searchLink fieldCode="JN" term="%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22">Proceedings of the National Academy of Sciences of the United States of America</searchLink>. 3/24/2026, Vol. 123 Issue 12, p1-11. 61p.
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  Data: <searchLink fieldCode="DE" term="%22Epimerization%22">Epimerization</searchLink><br /><searchLink fieldCode="DE" term="%22Electron+paramagnetic+resonance%22">Electron paramagnetic resonance</searchLink><br /><searchLink fieldCode="DE" term="%22Adenosylmethionine%22">Adenosylmethionine</searchLink><br /><searchLink fieldCode="DE" term="%22Peptide+synthesis%22">Peptide synthesis</searchLink><br /><searchLink fieldCode="DE" term="%22Ligand+binding+%28Biochemistry%29%22">Ligand binding (Biochemistry)</searchLink><br /><searchLink fieldCode="DE" term="%22Epimerases%22">Epimerases</searchLink><br /><searchLink fieldCode="DE" term="%22Natural+products%22">Natural products</searchLink>
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  Data: Radical S-adenosyl-l-methionine (SAM) enzymes figure prominently in the formation of ribosomally synthesized and posttranslationally modified peptides (RiPPs), where they catalyze peptide modifications including epimerization, thioether crosslink formation, and peptide backbone splicing. Here, we use rapid freeze-quench trapping together with electron paramagnetic resonance and electron-nuclear double resonance techniques to probe the mechanistic steps of the two epimerization reactions catalyzed by the radical SAM enzyme EpeE during conversion of its peptide substrate to the epipeptide natural product. Use of the EpeE C223S variant facilitated trapping and characterization of Ca radical intermediates, supporting a central role for C223 in the proposed epimerization mechanism. We showed that both wild-type and C223S EpeE with bound SAM and peptide substrate form the organometallic intermediate O upon reaction, and that thermal annealing of O results in conversion to an organic radical intermediate. Freeze-quenching at longer times allowed us to directly trap the organic radical intermediate, and isotopic labeling together with use of substrate variants allowed for detailed characterization of the substrate radical intermediates. The results revealed that while LC-MS enzymatic assays point to Ile12 as the initial site of epimerization, freeze-quench EPR reveals that Val4 is the preferred site for initial Ca radical formation. These apparently conflicting results were resolved by the observation that the Ile12 Ca radical is more efficiently quenched to form the d-Ile, thus providing insights into the determinants for substrate binding and epimerization by EpeE. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1073/pnas.2529780123
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        Text: English
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      – SubjectFull: Epimerization
        Type: general
      – SubjectFull: Electron paramagnetic resonance
        Type: general
      – SubjectFull: Adenosylmethionine
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      – SubjectFull: Peptide synthesis
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      – SubjectFull: Ligand binding (Biochemistry)
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      – SubjectFull: Epimerases
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      – SubjectFull: Natural products
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      – TitleFull: The radical SAM enzyme EpeE exhibits distinct site reactivity during the biosynthesis of the RiPP natural product epipeptide.
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            NameFull: Walls, William G.
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              Text: 3/24/2026
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              Y: 2026
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