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Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography
Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B(6)-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of sm...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10397204/ https://www.ncbi.nlm.nih.gov/pubmed/37532884 http://dx.doi.org/10.1038/s42004-023-00964-9 |
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author | Drago, Victoria N. Campos, Claudia Hooper, Mattea Collins, Aliyah Gerlits, Oksana Weiss, Kevin L. Blakeley, Matthew P. Phillips, Robert S. Kovalevsky, Andrey |
author_facet | Drago, Victoria N. Campos, Claudia Hooper, Mattea Collins, Aliyah Gerlits, Oksana Weiss, Kevin L. Blakeley, Matthew P. Phillips, Robert S. Kovalevsky, Andrey |
author_sort | Drago, Victoria N. |
collection | PubMed |
description | Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B(6)-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine. |
format | Online Article Text |
id | pubmed-10397204 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-103972042023-08-04 Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography Drago, Victoria N. Campos, Claudia Hooper, Mattea Collins, Aliyah Gerlits, Oksana Weiss, Kevin L. Blakeley, Matthew P. Phillips, Robert S. Kovalevsky, Andrey Commun Chem Article Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B(6)-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine. Nature Publishing Group UK 2023-08-03 /pmc/articles/PMC10397204/ /pubmed/37532884 http://dx.doi.org/10.1038/s42004-023-00964-9 Text en © This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Drago, Victoria N. Campos, Claudia Hooper, Mattea Collins, Aliyah Gerlits, Oksana Weiss, Kevin L. Blakeley, Matthew P. Phillips, Robert S. Kovalevsky, Andrey Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography |
title | Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography |
title_full | Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography |
title_fullStr | Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography |
title_full_unstemmed | Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography |
title_short | Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography |
title_sort | revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature x-ray and neutron crystallography |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10397204/ https://www.ncbi.nlm.nih.gov/pubmed/37532884 http://dx.doi.org/10.1038/s42004-023-00964-9 |
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