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dc.contributor.authorEly, Fernanda-
dc.contributor.authorNunes, José E.S.-
dc.contributor.authorSchroeder, Evelyn K.-
dc.contributor.authorFrazzon, Jeverson-
dc.contributor.authorPalma, Mario Sergio-
dc.contributor.authorSantos, Diógenes S.-
dc.contributor.authorBasso, Luiz A.-
dc.identifier.citationBMC Biochemistry, v. 9, n. 1, 2008.-
dc.description.abstractBackground. 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 FMN ox 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. © 2008 Ely et al; licensee BioMed Central Ltd.en
dc.subjectbacterial DNA-
dc.subjectchorismate synthase-
dc.subjectchorismic acid-
dc.subjectflavine mononucleotide reductase-
dc.subjectreduced nicotinamide adenine dinucleotide dehydrogenase-
dc.subjectshikimic acid-
dc.subjectflavine mononucleotide-
dc.subjectnicotinamide adenine dinucleotide-
dc.subjectprotein subunit-
dc.subjectDNA sequence-
dc.subjectenzyme analysis-
dc.subjectenzyme mechanism-
dc.subjectgel filtration chromatography-
dc.subjectgene amplification-
dc.subjectmass spectrometry-
dc.subjectmolecular cloning-
dc.subjectmolecular weight-
dc.subjectmultidrug resistance-
dc.subjectMycobacterium tuberculosis-
dc.subjectprotein expression-
dc.subjectprotein purification-
dc.subjectsolvent effect-
dc.subjectnucleotide sequence-
dc.subjectoxidation reduction reaction-
dc.subjectBacteria (microorganisms)-
dc.subjectBase Sequence-
dc.subjectChorismic Acid-
dc.subjectFlavin Mononucleotide-
dc.subjectPhosphorus-Oxygen Lyases-
dc.subjectProtein Subunits-
dc.titleThe Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthaseen
dc.contributor.institutionPontifícia Universidade Católica do Rio Grande do Sul (PUCRS)-
dc.contributor.institutionUniversidade Federal do Rio Grande do Sul (UFRGS)-
dc.contributor.institutionUniversidade Estadual Paulista (UNESP)-
dc.description.affiliationCentro de Pesquisas Em Biologia Molecular e Funcional Pontifícia Universidade Católica Do Rio Grande Do Sul, RS 90619-900, Porto Alegre-
dc.description.affiliationInstituto de Ciéncia e Tecnologia de Alimentos Universidade Federal Do Rio Grande Do Sul, RS 91501-970, Porto Alegre-
dc.description.affiliationDepartamento de Biologia/CEIS Universidade Estadual Paulista, SP 13506-900, Rio Claro-
dc.description.affiliationUnespDepartamento de Biologia/CEIS Universidade Estadual Paulista, SP 13506-900, Rio Claro-
dc.rights.accessRightsAcesso aberto-
dc.relation.ispartofBMC Biochemistry-
Appears in Collections:Artigos, TCCs, Teses e Dissertações da Unesp

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