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Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme
[Image: see text] Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet anothe...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical
Society
2017
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579537/ https://www.ncbi.nlm.nih.gov/pubmed/28768413 http://dx.doi.org/10.1021/jacs.7b04883 |
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author | Shisler, Krista A. Hutcheson, Rachel U. Horitani, Masaki Duschene, Kaitlin S. Crain, Adam V. Byer, Amanda S. Shepard, Eric M. Rasmussen, Ashley Yang, Jian Broderick, William E. Vey, Jessica L. Drennan, Catherine L. Hoffman, Brian M. Broderick, Joan B. |
author_facet | Shisler, Krista A. Hutcheson, Rachel U. Horitani, Masaki Duschene, Kaitlin S. Crain, Adam V. Byer, Amanda S. Shepard, Eric M. Rasmussen, Ashley Yang, Jian Broderick, William E. Vey, Jessica L. Drennan, Catherine L. Hoffman, Brian M. Broderick, Joan B. |
author_sort | Shisler, Krista A. |
collection | PubMed |
description | [Image: see text] Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B(12) enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na(+) as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by (23)Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M(+) ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[(13)C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li(+) to Cs(+), PFL-AE activity sharply maximizes at K(+), with NH(4)(+) closely matching the efficacy of K(+). PFL-AE is thus a type I M(+)-activated enzyme whose M(+) controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster. |
format | Online Article Text |
id | pubmed-5579537 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | American Chemical
Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-55795372017-09-05 Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme Shisler, Krista A. Hutcheson, Rachel U. Horitani, Masaki Duschene, Kaitlin S. Crain, Adam V. Byer, Amanda S. Shepard, Eric M. Rasmussen, Ashley Yang, Jian Broderick, William E. Vey, Jessica L. Drennan, Catherine L. Hoffman, Brian M. Broderick, Joan B. J Am Chem Soc [Image: see text] Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B(12) enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na(+) as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by (23)Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M(+) ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron–sulfur cluster. ENDOR studies of the PFL-AE/[(13)C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little–no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li(+) to Cs(+), PFL-AE activity sharply maximizes at K(+), with NH(4)(+) closely matching the efficacy of K(+). PFL-AE is thus a type I M(+)-activated enzyme whose M(+) controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron–sulfur cluster. American Chemical Society 2017-08-02 2017-08-30 /pmc/articles/PMC5579537/ /pubmed/28768413 http://dx.doi.org/10.1021/jacs.7b04883 Text en Copyright © 2017 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Shisler, Krista A. Hutcheson, Rachel U. Horitani, Masaki Duschene, Kaitlin S. Crain, Adam V. Byer, Amanda S. Shepard, Eric M. Rasmussen, Ashley Yang, Jian Broderick, William E. Vey, Jessica L. Drennan, Catherine L. Hoffman, Brian M. Broderick, Joan B. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme |
title | Monovalent
Cation Activation of the Radical SAM Enzyme
Pyruvate Formate-Lyase Activating Enzyme |
title_full | Monovalent
Cation Activation of the Radical SAM Enzyme
Pyruvate Formate-Lyase Activating Enzyme |
title_fullStr | Monovalent
Cation Activation of the Radical SAM Enzyme
Pyruvate Formate-Lyase Activating Enzyme |
title_full_unstemmed | Monovalent
Cation Activation of the Radical SAM Enzyme
Pyruvate Formate-Lyase Activating Enzyme |
title_short | Monovalent
Cation Activation of the Radical SAM Enzyme
Pyruvate Formate-Lyase Activating Enzyme |
title_sort | monovalent
cation activation of the radical sam enzyme
pyruvate formate-lyase activating enzyme |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579537/ https://www.ncbi.nlm.nih.gov/pubmed/28768413 http://dx.doi.org/10.1021/jacs.7b04883 |
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