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Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein
Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adeno...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8683613/ https://www.ncbi.nlm.nih.gov/pubmed/34801558 http://dx.doi.org/10.1016/j.jbc.2021.101423 |
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author | Andorfer, Mary C. Backman, Lindsey R.F. Li, Phoebe L. Ulrich, Emily C. Drennan, Catherine L. |
author_facet | Andorfer, Mary C. Backman, Lindsey R.F. Li, Phoebe L. Ulrich, Emily C. Drennan, Catherine L. |
author_sort | Andorfer, Mary C. |
collection | PubMed |
description | Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged. |
format | Online Article Text |
id | pubmed-8683613 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-86836132021-12-30 Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein Andorfer, Mary C. Backman, Lindsey R.F. Li, Phoebe L. Ulrich, Emily C. Drennan, Catherine L. J Biol Chem Research Article Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged. American Society for Biochemistry and Molecular Biology 2021-11-18 /pmc/articles/PMC8683613/ /pubmed/34801558 http://dx.doi.org/10.1016/j.jbc.2021.101423 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Andorfer, Mary C. Backman, Lindsey R.F. Li, Phoebe L. Ulrich, Emily C. Drennan, Catherine L. Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
title | Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
title_full | Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
title_fullStr | Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
title_full_unstemmed | Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
title_short | Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
title_sort | rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8683613/ https://www.ncbi.nlm.nih.gov/pubmed/34801558 http://dx.doi.org/10.1016/j.jbc.2021.101423 |
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