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Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, includ...

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Autores principales: Holmes, Jacob B., Liu, Viktoriia, Caulkins, Bethany G., Hilario, Eduardo, Ghosh, Rittik K., Drago, Victoria N., Young, Robert P., Romero, Jennifer A., Gill, Adam D., Bogie, Paul M., Paulino, Joana, Wang, Xiaoling, Riviere, Gwladys, Bosken, Yuliana K., Struppe, Jochem, Hassan, Alia, Guidoulianov, Jevgeni, Perrone, Barbara, Mentink-Vigier, Frederic, Chang, Chia-en A., Long, Joanna R., Hooley, Richard J., Mueser, Timothy C., Dunn, Michael F., Mueller, Leonard J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8764694/
https://www.ncbi.nlm.nih.gov/pubmed/34996869
http://dx.doi.org/10.1073/pnas.2109235119
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author Holmes, Jacob B.
Liu, Viktoriia
Caulkins, Bethany G.
Hilario, Eduardo
Ghosh, Rittik K.
Drago, Victoria N.
Young, Robert P.
Romero, Jennifer A.
Gill, Adam D.
Bogie, Paul M.
Paulino, Joana
Wang, Xiaoling
Riviere, Gwladys
Bosken, Yuliana K.
Struppe, Jochem
Hassan, Alia
Guidoulianov, Jevgeni
Perrone, Barbara
Mentink-Vigier, Frederic
Chang, Chia-en A.
Long, Joanna R.
Hooley, Richard J.
Mueser, Timothy C.
Dunn, Michael F.
Mueller, Leonard J.
author_facet Holmes, Jacob B.
Liu, Viktoriia
Caulkins, Bethany G.
Hilario, Eduardo
Ghosh, Rittik K.
Drago, Victoria N.
Young, Robert P.
Romero, Jennifer A.
Gill, Adam D.
Bogie, Paul M.
Paulino, Joana
Wang, Xiaoling
Riviere, Gwladys
Bosken, Yuliana K.
Struppe, Jochem
Hassan, Alia
Guidoulianov, Jevgeni
Perrone, Barbara
Mentink-Vigier, Frederic
Chang, Chia-en A.
Long, Joanna R.
Hooley, Richard J.
Mueser, Timothy C.
Dunn, Michael F.
Mueller, Leonard J.
author_sort Holmes, Jacob B.
collection PubMed
description NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C(β) and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.
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spelling pubmed-87646942022-01-26 Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate Holmes, Jacob B. Liu, Viktoriia Caulkins, Bethany G. Hilario, Eduardo Ghosh, Rittik K. Drago, Victoria N. Young, Robert P. Romero, Jennifer A. Gill, Adam D. Bogie, Paul M. Paulino, Joana Wang, Xiaoling Riviere, Gwladys Bosken, Yuliana K. Struppe, Jochem Hassan, Alia Guidoulianov, Jevgeni Perrone, Barbara Mentink-Vigier, Frederic Chang, Chia-en A. Long, Joanna R. Hooley, Richard J. Mueser, Timothy C. Dunn, Michael F. Mueller, Leonard J. Proc Natl Acad Sci U S A Biological Sciences NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C(β) and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does. National Academy of Sciences 2022-01-07 2022-01-11 /pmc/articles/PMC8764694/ /pubmed/34996869 http://dx.doi.org/10.1073/pnas.2109235119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Holmes, Jacob B.
Liu, Viktoriia
Caulkins, Bethany G.
Hilario, Eduardo
Ghosh, Rittik K.
Drago, Victoria N.
Young, Robert P.
Romero, Jennifer A.
Gill, Adam D.
Bogie, Paul M.
Paulino, Joana
Wang, Xiaoling
Riviere, Gwladys
Bosken, Yuliana K.
Struppe, Jochem
Hassan, Alia
Guidoulianov, Jevgeni
Perrone, Barbara
Mentink-Vigier, Frederic
Chang, Chia-en A.
Long, Joanna R.
Hooley, Richard J.
Mueser, Timothy C.
Dunn, Michael F.
Mueller, Leonard J.
Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate
title Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate
title_full Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate
title_fullStr Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate
title_full_unstemmed Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate
title_short Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate
title_sort imaging active site chemistry and protonation states: nmr crystallography of the tryptophan synthase α-aminoacrylate intermediate
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8764694/
https://www.ncbi.nlm.nih.gov/pubmed/34996869
http://dx.doi.org/10.1073/pnas.2109235119
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