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Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography
Haloalkane dehalogenase (HLD) enzymes employ an S(N)2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well‐characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we p...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley & Sons, Inc.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10503415/ https://www.ncbi.nlm.nih.gov/pubmed/37574754 http://dx.doi.org/10.1002/pro.4751 |
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author | Chmelova, Klaudia Gao, Tadeja Polak, Martin Schenkmayerova, Andrea Croll, Tristan I. Shaikh, Tanvir R. Skarupova, Jana Chaloupkova, Radka Diederichs, Kay Read, Randy J. Damborsky, Jiri Novacek, Jiri Marek, Martin |
author_facet | Chmelova, Klaudia Gao, Tadeja Polak, Martin Schenkmayerova, Andrea Croll, Tristan I. Shaikh, Tanvir R. Skarupova, Jana Chaloupkova, Radka Diederichs, Kay Read, Randy J. Damborsky, Jiri Novacek, Jiri Marek, Martin |
author_sort | Chmelova, Klaudia |
collection | PubMed |
description | Haloalkane dehalogenase (HLD) enzymes employ an S(N)2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well‐characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD‐like enzyme from the archaeon Haloferax mediterranei, by combining cryo‐electron microscopy (cryo‐EM) and x‐ray crystallography. We show that full‐length wild‐type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring‐like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three‐dimensional reconstructions of an oligomeric species by single‐particle cryo‐EM. Moreover, we engineered a crystallizable mutant (DhmeA(ΔGG)) that provided diffraction‐quality crystals. The 3.3 Å crystal structure reveals that DhmeA(ΔGG) forms a ring‐like 20‐mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates. |
format | Online Article Text |
id | pubmed-10503415 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | John Wiley & Sons, Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-105034152023-10-01 Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography Chmelova, Klaudia Gao, Tadeja Polak, Martin Schenkmayerova, Andrea Croll, Tristan I. Shaikh, Tanvir R. Skarupova, Jana Chaloupkova, Radka Diederichs, Kay Read, Randy J. Damborsky, Jiri Novacek, Jiri Marek, Martin Protein Sci Research Articles Haloalkane dehalogenase (HLD) enzymes employ an S(N)2 nucleophilic substitution mechanism to erase halogen substituents in diverse organohalogen compounds. Subfamily I and II HLDs are well‐characterized enzymes, but the mode and purpose of multimerization of subfamily III HLDs are unknown. Here we probe the structural organization of DhmeA, a subfamily III HLD‐like enzyme from the archaeon Haloferax mediterranei, by combining cryo‐electron microscopy (cryo‐EM) and x‐ray crystallography. We show that full‐length wild‐type DhmeA forms diverse quaternary structures, ranging from small oligomers to large supramolecular ring‐like assemblies of various sizes and symmetries. We optimized sample preparation steps, enabling three‐dimensional reconstructions of an oligomeric species by single‐particle cryo‐EM. Moreover, we engineered a crystallizable mutant (DhmeA(ΔGG)) that provided diffraction‐quality crystals. The 3.3 Å crystal structure reveals that DhmeA(ΔGG) forms a ring‐like 20‐mer structure with outer and inner diameter of ~200 and ~80 Å, respectively. An enzyme homodimer represents a basic repeating building unit of the crystallographic ring. Three assembly interfaces (dimerization, tetramerization, and multimerization) were identified to form the supramolecular ring that displays a negatively charged exterior, while its interior part harboring catalytic sites is positively charged. Localization and exposure of catalytic machineries suggest a possible processing of large negatively charged macromolecular substrates. John Wiley & Sons, Inc. 2023-10-01 /pmc/articles/PMC10503415/ /pubmed/37574754 http://dx.doi.org/10.1002/pro.4751 Text en © 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Chmelova, Klaudia Gao, Tadeja Polak, Martin Schenkmayerova, Andrea Croll, Tristan I. Shaikh, Tanvir R. Skarupova, Jana Chaloupkova, Radka Diederichs, Kay Read, Randy J. Damborsky, Jiri Novacek, Jiri Marek, Martin Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography |
title | Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography |
title_full | Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography |
title_fullStr | Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography |
title_full_unstemmed | Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography |
title_short | Multimeric structure of a subfamily III haloalkane dehalogenase‐like enzyme solved by combination of cryo‐EM and x‐ray crystallography |
title_sort | multimeric structure of a subfamily iii haloalkane dehalogenase‐like enzyme solved by combination of cryo‐em and x‐ray crystallography |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10503415/ https://www.ncbi.nlm.nih.gov/pubmed/37574754 http://dx.doi.org/10.1002/pro.4751 |
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