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Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy
Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH(3)-H(4)folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S-adenosylmethionine with the folate cycle in one-carbon metabolis...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Cold Spring Harbor Laboratory
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9934640/ https://www.ncbi.nlm.nih.gov/pubmed/36798380 http://dx.doi.org/10.1101/2023.02.11.528079 |
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author | Watkins, Maxwell B. Wang, Haoyue Burnim, Audrey Ando, Nozomi |
author_facet | Watkins, Maxwell B. Wang, Haoyue Burnim, Audrey Ando, Nozomi |
author_sort | Watkins, Maxwell B. |
collection | PubMed |
description | Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH(3)-H(4)folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multi-domain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a “divide and conquer” approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryo-electron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present the first structural description of MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH(3)-H(4)folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH. |
format | Online Article Text |
id | pubmed-9934640 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Cold Spring Harbor Laboratory |
record_format | MEDLINE/PubMed |
spelling | pubmed-99346402023-02-17 Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy Watkins, Maxwell B. Wang, Haoyue Burnim, Audrey Ando, Nozomi bioRxiv Article Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH(3)-H(4)folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multi-domain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a “divide and conquer” approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryo-electron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present the first structural description of MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH(3)-H(4)folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH. Cold Spring Harbor Laboratory 2023-02-12 /pmc/articles/PMC9934640/ /pubmed/36798380 http://dx.doi.org/10.1101/2023.02.11.528079 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator. |
spellingShingle | Article Watkins, Maxwell B. Wang, Haoyue Burnim, Audrey Ando, Nozomi Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy |
title | Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy |
title_full | Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy |
title_fullStr | Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy |
title_full_unstemmed | Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy |
title_short | Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy |
title_sort | conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle x-ray scattering and cryo-electron microscopy |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9934640/ https://www.ncbi.nlm.nih.gov/pubmed/36798380 http://dx.doi.org/10.1101/2023.02.11.528079 |
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