Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/134441 |
Resumo: | Mo/W-Formate dehydrogenases (Fdhs) are unique bacterial enzymes that catalyze the reversible reduction of CO2 to formate. This capability is not just a possible route for greenhouse gas sequestration, but also a possibility to create a compound that is currently used as a fuel. Only five different Fdhs have their structure solved and the two recent structures of FdhAB from Desulfovibrio vulgaris (oxidized and formate-reduced forms) showed conformational changes that may bring new insights about the catalytic mechanism. So, the main goals of this work are to attain new structures of reduced forms of FdhAB wild type and the new variant FdhAB-C872A by X-ray crystallography. Furthermore, we optimized the crystallization conditions to produce FdhAB microcrystals that will be used in serial crystallography and time resolved experiments. We crystallized FdhAB reduced with sodium dithionite and compared with the previously published formate-reduced form (PDB CODE: 6SDV). The structure of dithionite-reduced form of FdhAB was obtained by soaking the crystal with sodium dithionite and the crystals diffracted beyond 1.53 Å (the Fdh model at higher resolution). The active sites of the new dithionite-reduced structure and formate-reduced structure were then compared, and it was concluded that there are no significant differences between these two reduced states. To study the role of the conserved disulphide bridge (C845-C872, D. vulgaris numbering) in some Fdhs from Desulfovibrio, different FdhAB variants were produced by our collaborators, and we were able to crystallize and obtain different structures of the new variant FdhAB-C872A in aerobic and anaerobic conditions. The first structure of Fdh-C872A variant was obtained in aerobic conditions at a resolution of 2.27 Å. In anaerobic conditions, we obtained two good crystals of Fdh-C872A cocrystallized with sodium azide and sodium dithionite that diffracted beyond 2.4 Å 2.8 Å, respectively. The three structures of the Fdh-C872A variant are very similar, however, the active site seems to lose the sulphide ligand when the enzyme is exposed to oxygen. Comparing the active site of Fdh-C872A variant with the previous structures (PDB CODES: 6SDV and 6SDR) we concluded that it presents several differences that are the result of significant rearrangements in part of the polypeptide chain of the catalytic subunit, promoted by the disulphide bridge break. Finally, we optimized the conditions to produce micro-crystals for serial crystallography and tine resolved experiments. Several conditions were tested for FdhAB crystallization by using the crystallization robot that allowed us to plot the phases diagrams. The steps of optimization and scale-up were also successful, considering that the micro-crystals appeared, and the crystals features were reproducible. But when we tried to evaluate the crystal diffraction power in an synchrotron source, the preliminary diffraction results showed a maximum observed resolution of about 2.9 Å with very few hits. So, it was concluded that further optimization of crystallization conditions is required. |
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Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixationFormate dehydrogenasesreduction of CO2formateX-ray crystallographyserial crystallography and tine resolved experimentsDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasMo/W-Formate dehydrogenases (Fdhs) are unique bacterial enzymes that catalyze the reversible reduction of CO2 to formate. This capability is not just a possible route for greenhouse gas sequestration, but also a possibility to create a compound that is currently used as a fuel. Only five different Fdhs have their structure solved and the two recent structures of FdhAB from Desulfovibrio vulgaris (oxidized and formate-reduced forms) showed conformational changes that may bring new insights about the catalytic mechanism. So, the main goals of this work are to attain new structures of reduced forms of FdhAB wild type and the new variant FdhAB-C872A by X-ray crystallography. Furthermore, we optimized the crystallization conditions to produce FdhAB microcrystals that will be used in serial crystallography and time resolved experiments. We crystallized FdhAB reduced with sodium dithionite and compared with the previously published formate-reduced form (PDB CODE: 6SDV). The structure of dithionite-reduced form of FdhAB was obtained by soaking the crystal with sodium dithionite and the crystals diffracted beyond 1.53 Å (the Fdh model at higher resolution). The active sites of the new dithionite-reduced structure and formate-reduced structure were then compared, and it was concluded that there are no significant differences between these two reduced states. To study the role of the conserved disulphide bridge (C845-C872, D. vulgaris numbering) in some Fdhs from Desulfovibrio, different FdhAB variants were produced by our collaborators, and we were able to crystallize and obtain different structures of the new variant FdhAB-C872A in aerobic and anaerobic conditions. The first structure of Fdh-C872A variant was obtained in aerobic conditions at a resolution of 2.27 Å. In anaerobic conditions, we obtained two good crystals of Fdh-C872A cocrystallized with sodium azide and sodium dithionite that diffracted beyond 2.4 Å 2.8 Å, respectively. The three structures of the Fdh-C872A variant are very similar, however, the active site seems to lose the sulphide ligand when the enzyme is exposed to oxygen. Comparing the active site of Fdh-C872A variant with the previous structures (PDB CODES: 6SDV and 6SDR) we concluded that it presents several differences that are the result of significant rearrangements in part of the polypeptide chain of the catalytic subunit, promoted by the disulphide bridge break. Finally, we optimized the conditions to produce micro-crystals for serial crystallography and tine resolved experiments. Several conditions were tested for FdhAB crystallization by using the crystallization robot that allowed us to plot the phases diagrams. The steps of optimization and scale-up were also successful, considering that the micro-crystals appeared, and the crystals features were reproducible. But when we tried to evaluate the crystal diffraction power in an synchrotron source, the preliminary diffraction results showed a maximum observed resolution of about 2.9 Å with very few hits. So, it was concluded that further optimization of crystallization conditions is required.As Formato desidrogenases de Mo ou W (Fdhs) são enzimas bacterianas únicas que catalisam a redução reversível de CO2 a formato. Esta capacidade além de possibilitar a captura de gases de efeito estufa, oferece a possibilidade de criar um composto que pode ser usado como combustível. Apenas cinco Fdhs têm as suas estruturas moleculares determinadas e as duas recentes estruturas de FdhAB de Desulfovibrio vulgaris (formas oxidada e reduzida) mostraram alterações conformacionais que trazem novas perceções sobre o mecanismo catalítico. Assim, os principais objetivos deste trabalho são obter novas estruturas de formas reduzidas de FdhAB wild type e de uma nova variante, FdhAB-C872A, por cristalografia de raios-X. Além disso, otimizamos as condições de cristalização para produzir microcristais de FdhAB que serão usados em experiências de cristalografia em série e de resolução no tempo. Nós cristalizámos FdhAB reduzido com ditionito de sódio e comparámos com a forma reduzida com formato, publicado anteriormente (CÓDIGO PDB: 6SDV). A estrutura da forma reduzida com ditionito foi obtida por incubação dos cristais com ditionito de sódio e os cristais difrataram a 1,53 Å (tornando-se na estrutura de Fdh resolvida a mais alta resolução). Os centros ativos da nova estrutura reduzida com ditionito e da estrutura reduzida com formato foram depois comparados e concluiu-se que não há diferenças significativas entre estes dois estados reduzidos. Para estudar o papel da ponte dissulfureto conservada (C845-C872, numeração de D. vulgaris) em algumas Fdhs de Desulfovibrio, diferentes variantes de FdhAB foram produzidas pelos nossos colaboradores, e nós conseguimos cristalizar e obter as estruturas duma nova variante, FdhAB-C872A, em condições aeróbias e anaeróbias. A primeira estrutura da variante Fdh-C872A em condições aeróbias foi obtida a uma resolução de 2.27 Å. Em condições anaeróbias nós obtivemos dois cristais de boa qualidade de FdhAB-C872A co-cristalizada com azida de sódio e com ditionito de sódio que difrataram a 2.4 Å e 2.8 Å, respetivamente. As três estruturas da variante Fdh-C872A são muito semelhantes, no entanto, o centro ativo parece perder o ligando de enxofre quando a enzima é exposta ao oxigênio. Comparando o centro ativo da variante Fdh-C872A com as estruturas anteriores (CÓDIGOS PDB: 6SDV e 6SDR) nós concluímos que esta apresenta várias diferenças que são o resultado de rearranjos significativos em parte da cadeia polipeptídica da subunidade catalítica, promovida pela quebra da ponte dissulfureto. Por fim, otimizámos as condições de produção de microcristais para experiências de cristalografia em série e de resolução no tempo. Várias condições foram testadas para a cristalização de FdhAB usando o robô de cristalização, que nos permitiu traçar o diagramas das fases. As etapas de otimização e escalonamento também foram conseguidas, visto que os microcristais apareceram em volumes maiores e as suas características eram reprodutíveis. No entanto, quando tentámos avaliar o poder de difração dos cristais numa fonte de sincrotrão, os resultados preliminares da difração mostraram uma resolução máxima observada de cerca de 2.9 Å. Assim, concluiu-se que estas condições de cristalização ainda precisam duma otimização posterior.Mota, CristianoRomão, Maria JoãoRUNKlymanska, Kateryna2022-02-232024-12-01T00:00:00Z2022-02-23T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/134441enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:12:52Zoai:run.unl.pt:10362/134441Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:48:05.376920Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
title |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
spellingShingle |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation Klymanska, Kateryna Formate dehydrogenases reduction of CO2 formate X-ray crystallography serial crystallography and tine resolved experiments Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
title_full |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
title_fullStr |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
title_full_unstemmed |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
title_sort |
Structural studies of a W-Formate dehydrogenase by X-ray crystallography – a bacterial enzyme that catalyze greenhouse gas (CO2) fixation |
author |
Klymanska, Kateryna |
author_facet |
Klymanska, Kateryna |
author_role |
author |
dc.contributor.none.fl_str_mv |
Mota, Cristiano Romão, Maria João RUN |
dc.contributor.author.fl_str_mv |
Klymanska, Kateryna |
dc.subject.por.fl_str_mv |
Formate dehydrogenases reduction of CO2 formate X-ray crystallography serial crystallography and tine resolved experiments Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
Formate dehydrogenases reduction of CO2 formate X-ray crystallography serial crystallography and tine resolved experiments Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
Mo/W-Formate dehydrogenases (Fdhs) are unique bacterial enzymes that catalyze the reversible reduction of CO2 to formate. This capability is not just a possible route for greenhouse gas sequestration, but also a possibility to create a compound that is currently used as a fuel. Only five different Fdhs have their structure solved and the two recent structures of FdhAB from Desulfovibrio vulgaris (oxidized and formate-reduced forms) showed conformational changes that may bring new insights about the catalytic mechanism. So, the main goals of this work are to attain new structures of reduced forms of FdhAB wild type and the new variant FdhAB-C872A by X-ray crystallography. Furthermore, we optimized the crystallization conditions to produce FdhAB microcrystals that will be used in serial crystallography and time resolved experiments. We crystallized FdhAB reduced with sodium dithionite and compared with the previously published formate-reduced form (PDB CODE: 6SDV). The structure of dithionite-reduced form of FdhAB was obtained by soaking the crystal with sodium dithionite and the crystals diffracted beyond 1.53 Å (the Fdh model at higher resolution). The active sites of the new dithionite-reduced structure and formate-reduced structure were then compared, and it was concluded that there are no significant differences between these two reduced states. To study the role of the conserved disulphide bridge (C845-C872, D. vulgaris numbering) in some Fdhs from Desulfovibrio, different FdhAB variants were produced by our collaborators, and we were able to crystallize and obtain different structures of the new variant FdhAB-C872A in aerobic and anaerobic conditions. The first structure of Fdh-C872A variant was obtained in aerobic conditions at a resolution of 2.27 Å. In anaerobic conditions, we obtained two good crystals of Fdh-C872A cocrystallized with sodium azide and sodium dithionite that diffracted beyond 2.4 Å 2.8 Å, respectively. The three structures of the Fdh-C872A variant are very similar, however, the active site seems to lose the sulphide ligand when the enzyme is exposed to oxygen. Comparing the active site of Fdh-C872A variant with the previous structures (PDB CODES: 6SDV and 6SDR) we concluded that it presents several differences that are the result of significant rearrangements in part of the polypeptide chain of the catalytic subunit, promoted by the disulphide bridge break. Finally, we optimized the conditions to produce micro-crystals for serial crystallography and tine resolved experiments. Several conditions were tested for FdhAB crystallization by using the crystallization robot that allowed us to plot the phases diagrams. The steps of optimization and scale-up were also successful, considering that the micro-crystals appeared, and the crystals features were reproducible. But when we tried to evaluate the crystal diffraction power in an synchrotron source, the preliminary diffraction results showed a maximum observed resolution of about 2.9 Å with very few hits. So, it was concluded that further optimization of crystallization conditions is required. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-02-23 2022-02-23T00:00:00Z 2024-12-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
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http://hdl.handle.net/10362/134441 |
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http://hdl.handle.net/10362/134441 |
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eng |
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