The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase
Autor(a) principal: | |
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Data de Publicação: | 2008 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFRGS |
Texto Completo: | http://hdl.handle.net/10183/21543 |
Resumo: | Background: The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results: In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion: This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. |
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Ely, FernandaNunes, José Eduardo SacconiSchroeder, Evelyn KoecheFrazzon, JeversonPalma, Mário SérgioSantos, Diogenes SantiagoBasso, Luiz Augusto2010-05-05T04:15:47Z2008http://hdl.handle.net/10183/21543000639072Background: The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results: In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion: This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.application/pdfengBMC biochemistry. London. vol. 9, n. 3 (apr. 2008), p. 1-16Mycobacterium tuberculosisThe Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthaseEstrangeiroinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFRGSinstname:Universidade Federal do Rio Grande do Sul (UFRGS)instacron:UFRGSORIGINAL000639072.pdf000639072.pdfTexto completo (inglês)application/pdf578340http://www.lume.ufrgs.br/bitstream/10183/21543/1/000639072.pdff45f90cd8be577d950920273b79cd9c3MD51TEXT000639072.pdf.txt000639072.pdf.txtExtracted Texttext/plain61063http://www.lume.ufrgs.br/bitstream/10183/21543/2/000639072.pdf.txtfae7425be7cd6a805808706640467333MD52THUMBNAIL000639072.pdf.jpg000639072.pdf.jpgGenerated Thumbnailimage/jpeg2089http://www.lume.ufrgs.br/bitstream/10183/21543/3/000639072.pdf.jpgcc773c1188668f303bd8d8cc65bcb4bfMD5310183/215432021-06-13 04:32:19.158097oai:www.lume.ufrgs.br:10183/21543Repositório de PublicaçõesPUBhttps://lume.ufrgs.br/oai/requestopendoar:2021-06-13T07:32:19Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS)false |
dc.title.pt_BR.fl_str_mv |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
title |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
spellingShingle |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase Ely, Fernanda Mycobacterium tuberculosis |
title_short |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
title_full |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
title_fullStr |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
title_full_unstemmed |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
title_sort |
The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
author |
Ely, Fernanda |
author_facet |
Ely, Fernanda Nunes, José Eduardo Sacconi Schroeder, Evelyn Koeche Frazzon, Jeverson Palma, Mário Sérgio Santos, Diogenes Santiago Basso, Luiz Augusto |
author_role |
author |
author2 |
Nunes, José Eduardo Sacconi Schroeder, Evelyn Koeche Frazzon, Jeverson Palma, Mário Sérgio Santos, Diogenes Santiago Basso, Luiz Augusto |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Ely, Fernanda Nunes, José Eduardo Sacconi Schroeder, Evelyn Koeche Frazzon, Jeverson Palma, Mário Sérgio Santos, Diogenes Santiago Basso, Luiz Augusto |
dc.subject.por.fl_str_mv |
Mycobacterium tuberculosis |
topic |
Mycobacterium tuberculosis |
description |
Background: The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results: In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion: This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. |
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2008 |
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BMC biochemistry. London. vol. 9, n. 3 (apr. 2008), p. 1-16 |
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