A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico
Ano de defesa: | 2013 |
---|---|
Autor(a) principal: | |
Orientador(a): | |
Banca de defesa: | |
Tipo de documento: | Tese |
Tipo de acesso: | Acesso aberto |
Idioma: | por |
Instituição de defesa: |
Pontif?cia Universidade Cat?lica do Rio Grande do Sul
|
Programa de Pós-Graduação: |
Programa de P?s-Gradua??o em Biologia Celular e Molecular
|
Departamento: |
Faculdade de Bioci?ncias
|
País: |
BR
|
Palavras-chave em Português: | |
Área do conhecimento CNPq: | |
Link de acesso: | http://tede2.pucrs.br/tede2/handle/tede/5473 |
Resumo: | Human tuberculosis (TB) is a major global health threat. The disease s increasing prevalence, coupled with the emergence of drug-resistant strains and the devastating effects of co-infection with human immunodeficiency virus (HIV) indicate an urgent need for the development of new and more efficient drugs to combat the disease's causative agent, Mycobacterium tuberculosis. Global health authorities have also begun to recognize the need for drugs that can kill the mycobacteria in its different physiological states including but not limited to latent TB infection. A more comprehensive understanding of the bacilli's nucleotide metabolic pathways, in particular purine and pyrimidine salvage pathways, could aid in the development of new therapeutic strategies to reduce the incidence of TB worldwide. Nucleoside hydrolase catalyzes the irreversible hydrolysis of N-glycosidic bond of ribonucleosides, forming α-D-ribose and the corresponding base. This work describes the amplification, cloning, expression and purification of the iunH-encoding purine nucleoside hydrolase from M. tuberculosis (MtIAGU-NH). Results from steadystate kinetic experiments indicate that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Kinetics analysis utilizing fluorescence spectroscopy of ribose binding to MtIAGU-NH suggests that prior to ligand association there are two pre-existing forms of the enzyme. We determined the intracellular concentrations of inosine, uridine, hypoxanthine, and uracil as well as the reaction s thermodynamic parameters. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the MtIAGU-NH-catalyzed chemical reaction, along with results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiments, multiple sequence alignment, and molecular docking experiments are also presented. Knockout experiments of the iunH gene indicate that this gene is not essential for the growth of M. tuberculosis H37Rv underutilized experimental conditions. The data presented here contribute to our understanding of MtIAGU-NH, providing a solid basis for the development of efficient prophylactic and therapeutic strategies to reduce the global incidence of TB. |
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Santos, Di?genes SantiagoCPF:18729258804http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4721461Z7CPF:00792297008http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4550356Y0Wink, Priscila Lamb2015-04-14T14:51:28Z2013-10-072013-07-30WINK, Priscila Lamb. A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico. 2013. 100 f. Tese (Doutorado em Biologia Celular e Molecular) - Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Porto Alegre, 2013.http://tede2.pucrs.br/tede2/handle/tede/5473Human tuberculosis (TB) is a major global health threat. The disease s increasing prevalence, coupled with the emergence of drug-resistant strains and the devastating effects of co-infection with human immunodeficiency virus (HIV) indicate an urgent need for the development of new and more efficient drugs to combat the disease's causative agent, Mycobacterium tuberculosis. Global health authorities have also begun to recognize the need for drugs that can kill the mycobacteria in its different physiological states including but not limited to latent TB infection. A more comprehensive understanding of the bacilli's nucleotide metabolic pathways, in particular purine and pyrimidine salvage pathways, could aid in the development of new therapeutic strategies to reduce the incidence of TB worldwide. Nucleoside hydrolase catalyzes the irreversible hydrolysis of N-glycosidic bond of ribonucleosides, forming α-D-ribose and the corresponding base. This work describes the amplification, cloning, expression and purification of the iunH-encoding purine nucleoside hydrolase from M. tuberculosis (MtIAGU-NH). Results from steadystate kinetic experiments indicate that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Kinetics analysis utilizing fluorescence spectroscopy of ribose binding to MtIAGU-NH suggests that prior to ligand association there are two pre-existing forms of the enzyme. We determined the intracellular concentrations of inosine, uridine, hypoxanthine, and uracil as well as the reaction s thermodynamic parameters. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the MtIAGU-NH-catalyzed chemical reaction, along with results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiments, multiple sequence alignment, and molecular docking experiments are also presented. Knockout experiments of the iunH gene indicate that this gene is not essential for the growth of M. tuberculosis H37Rv underutilized experimental conditions. The data presented here contribute to our understanding of MtIAGU-NH, providing a solid basis for the development of efficient prophylactic and therapeutic strategies to reduce the global incidence of TB.A tuberculose humana (TB) ? considerada uma amea?a ? sa?de global. O aumento na preval?ncia da doen?a, a emerg?ncia de cepas resistentes e a coinfec??o com o v?rus da imunodefici?ncia humana levaram ? urgente necessidade do desenvolvimento de novas drogas mais eficientes para o combate do agente causativo da TB, Mycobacterium tuberculosis. Al?m disso, h? a necessidade de novas drogas contra a micobact?ria nos seus diferentes estados fisiol?gicos, incluindo a TB latente. Um maior entendimento sobre as vias metab?licas de nucleot?deos do bacilo da TB, em particular as vias de salvamento de purinas e pirimidinas, levariam ao desenvolvimento de novas estrat?gias terap?uticas para controlar a incid?ncia global de TB. A hidrolase de nucleos?deos catalisa a hidr?lise irrevers?vel da liga??o N-glicos?dica de ribonucleos?deos, dando origem ? α-D-ribose e sua base livre correspondente. Este trabalho descreve a amplifica??o e clonagem do gene iunH, e a express?o e purifica??o da enzima recombinante hidrolase de nucleos?deos pur?nicos de M. tuberculosis (MtIAGU-NH). Os resultados de cin?tica no estado estacion?rio mostram que a MtIAGU-NH possui uma ampla especificidade pelos substratos, utilizando inosina, adenosina, guanosina e uridina como substratos. As medidas cin?ticas da liga??o da ribose ? MtIAGU-NH por espectroscopia fluorim?trica sugerem a exist?ncia de duas formas da enzima em equil?brio, relacionadas ? associa??o ao ligante. As concentra??es intracelulares de inosina, uridina, hipoxantina e uracil foram determinadas e os par?metros termodin?micos, estimados. Os par?metros termodin?micos de ativa??o (Ea, ΔG#, ΔS#, ΔH#) para a rea??o qu?mica catalisada pela MtIAGU-NH, al?m dos resultados de espectrometria de massa, calorimetria de titula??o isot?rmica, experimento de perfil de pH, alinhamento de m?ltiplas sequ?ncias e experimentos de docagem molecular tamb?m s?o apresentados neste trabalho. O nocaute do gene iunH mostrou que este n?o ? essencial para o crescimento do bacilo M. tuberculosis H37Rv nas condi??es experimentais empregadas. Os resultados aqui descritos dever?o ser ?teis para um melhor entendimento sobre a enzima MtIAGU-NH, fornecendo bases s?lidas para o desenvolvimento de estrat?gias terap?uticas e preventivas para diminuir a incid?ncia global da TB.Made available in DSpace on 2015-04-14T14:51:28Z (GMT). No. of bitstreams: 1 451400.pdf: 16861193 bytes, checksum: 8e59e1dc262f6d66b6ff62e7eca281b9 (MD5) Previous issue date: 2013-07-30application/pdfhttp://tede2.pucrs.br:80/tede2/retrieve/16440/451400.pdf.jpgporPontif?cia Universidade Cat?lica do Rio Grande do SulPrograma de P?s-Gradua??o em Biologia Celular e MolecularPUCRSBRFaculdade de Bioci?nciasBIOLOGIA MOLECULARBIOLOGIA CELULARTUBERCULOSEENZIMASCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARA enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nicoinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis819824693009663736060060036528317262667714info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da PUC_RSinstname:Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)instacron:PUC_RSTHUMBNAIL451400.pdf.jpg451400.pdf.jpgimage/jpeg3376http://tede2.pucrs.br/tede2/bitstream/tede/5473/3/451400.pdf.jpg55d8a3216a9eee50088d14aaade585f2MD53TEXT451400.pdf.txt451400.pdf.txttext/plain174107http://tede2.pucrs.br/tede2/bitstream/tede/5473/2/451400.pdf.txt2badb3a087092a834d182f41053f7462MD52ORIGINAL451400.pdfapplication/pdf16861193http://tede2.pucrs.br/tede2/bitstream/tede/5473/1/451400.pdf8e59e1dc262f6d66b6ff62e7eca281b9MD51tede/54732015-05-14 11:40:29.968oai:tede2.pucrs.br:tede/5473Biblioteca Digital de Teses e Dissertaçõeshttp://tede2.pucrs.br/tede2/PRIhttps://tede2.pucrs.br/oai/requestbiblioteca.central@pucrs.br||opendoar:2015-05-14T14:40:29Biblioteca Digital de Teses e Dissertações da PUC_RS - Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS)false |
dc.title.por.fl_str_mv |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
title |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
spellingShingle |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico Wink, Priscila Lamb BIOLOGIA MOLECULAR BIOLOGIA CELULAR TUBERCULOSE ENZIMAS CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
title_short |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
title_full |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
title_fullStr |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
title_full_unstemmed |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
title_sort |
A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico |
author |
Wink, Priscila Lamb |
author_facet |
Wink, Priscila Lamb |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Santos, Di?genes Santiago |
dc.contributor.advisor1ID.fl_str_mv |
CPF:18729258804 |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4721461Z7 |
dc.contributor.authorID.fl_str_mv |
CPF:00792297008 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4550356Y0 |
dc.contributor.author.fl_str_mv |
Wink, Priscila Lamb |
contributor_str_mv |
Santos, Di?genes Santiago |
dc.subject.por.fl_str_mv |
BIOLOGIA MOLECULAR BIOLOGIA CELULAR TUBERCULOSE ENZIMAS |
topic |
BIOLOGIA MOLECULAR BIOLOGIA CELULAR TUBERCULOSE ENZIMAS CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR |
description |
Human tuberculosis (TB) is a major global health threat. The disease s increasing prevalence, coupled with the emergence of drug-resistant strains and the devastating effects of co-infection with human immunodeficiency virus (HIV) indicate an urgent need for the development of new and more efficient drugs to combat the disease's causative agent, Mycobacterium tuberculosis. Global health authorities have also begun to recognize the need for drugs that can kill the mycobacteria in its different physiological states including but not limited to latent TB infection. A more comprehensive understanding of the bacilli's nucleotide metabolic pathways, in particular purine and pyrimidine salvage pathways, could aid in the development of new therapeutic strategies to reduce the incidence of TB worldwide. Nucleoside hydrolase catalyzes the irreversible hydrolysis of N-glycosidic bond of ribonucleosides, forming α-D-ribose and the corresponding base. This work describes the amplification, cloning, expression and purification of the iunH-encoding purine nucleoside hydrolase from M. tuberculosis (MtIAGU-NH). Results from steadystate kinetic experiments indicate that MtIAGU-NH has broad substrate specificity, accepting inosine, adenosine, guanosine, and uridine as substrates. Kinetics analysis utilizing fluorescence spectroscopy of ribose binding to MtIAGU-NH suggests that prior to ligand association there are two pre-existing forms of the enzyme. We determined the intracellular concentrations of inosine, uridine, hypoxanthine, and uracil as well as the reaction s thermodynamic parameters. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the MtIAGU-NH-catalyzed chemical reaction, along with results from mass spectrometry, isothermal titration calorimetry (ITC), pH-rate profile experiments, multiple sequence alignment, and molecular docking experiments are also presented. Knockout experiments of the iunH gene indicate that this gene is not essential for the growth of M. tuberculosis H37Rv underutilized experimental conditions. The data presented here contribute to our understanding of MtIAGU-NH, providing a solid basis for the development of efficient prophylactic and therapeutic strategies to reduce the global incidence of TB. |
publishDate |
2013 |
dc.date.available.fl_str_mv |
2013-10-07 |
dc.date.issued.fl_str_mv |
2013-07-30 |
dc.date.accessioned.fl_str_mv |
2015-04-14T14:51:28Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
WINK, Priscila Lamb. A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico. 2013. 100 f. Tese (Doutorado em Biologia Celular e Molecular) - Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Porto Alegre, 2013. |
dc.identifier.uri.fl_str_mv |
http://tede2.pucrs.br/tede2/handle/tede/5473 |
identifier_str_mv |
WINK, Priscila Lamb. A enzima hidrolase de nucleos?deos pur?nicos de Mycobacterium tuberculosis H37RV : caracteriza??o bioqu?mica, termodin?mica e estudos de nocaute g?nico. 2013. 100 f. Tese (Doutorado em Biologia Celular e Molecular) - Pontif?cia Universidade Cat?lica do Rio Grande do Sul, Porto Alegre, 2013. |
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http://tede2.pucrs.br/tede2/handle/tede/5473 |
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Pontif?cia Universidade Cat?lica do Rio Grande do Sul |
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Pontif?cia Universidade Cat?lica do Rio Grande do Sul |
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