The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

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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 [UNESP]
dc.contributor.authorSantos, Diógenes S.
dc.contributor.authorBasso, Luiz A.
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.date.accessioned2014-05-27T11:23:33Z
dc.date.available2014-05-27T11:23:33Z
dc.date.issued2008-05-22
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.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.identifierhttp://dx.doi.org/10.1186/1471-2091-9-13
dc.identifier.citationBMC Biochemistry, v. 9, n. 1, 2008.
dc.identifier.doi10.1186/1471-2091-9-13
dc.identifier.file2-s2.0-43749101877.pdf
dc.identifier.issn1471-2091
dc.identifier.lattes2901888624506535
dc.identifier.scopus2-s2.0-43749101877
dc.identifier.urihttp://hdl.handle.net/11449/70417
dc.language.isoeng
dc.relation.ispartofBMC Biochemistry
dc.relation.ispartofjcr1.595
dc.relation.ispartofsjr0,708
dc.rights.accessRightsAcesso aberto
dc.sourceScopus
dc.subjectbacterial DNA
dc.subjectchorismate synthase
dc.subjectchorismic acid
dc.subjectflavine mononucleotide reductase
dc.subjectreduced nicotinamide adenine dinucleotide dehydrogenase
dc.subjectshikimic acid
dc.subjectsynthetase
dc.subjectflavine mononucleotide
dc.subjectlyase
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.subjectnonhuman
dc.subjectprotein expression
dc.subjectprotein purification
dc.subjectsolvent effect
dc.subjectspectrofluorometry
dc.subjectspectrophotometry
dc.subjectbiosynthesis
dc.subjectcatalysis
dc.subjectchemistry
dc.subjectenzymology
dc.subjectgenetics
dc.subjectmetabolism
dc.subjectnucleotide sequence
dc.subjectoxidation reduction reaction
dc.subjectBacteria (microorganisms)
dc.subjectBase Sequence
dc.subjectCatalysis
dc.subjectChorismic Acid
dc.subjectFlavin Mononucleotide
dc.subjectNAD
dc.subjectOxidation-Reduction
dc.subjectPhosphorus-Oxygen Lyases
dc.subjectProtein Subunits
dc.titleThe Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthaseen
dc.typeArtigo
dcterms.licensehttp://www.biomedcentral.com/about/license
unesp.author.lattes2901888624506535
unesp.campusUniversidade Estadual Paulista (Unesp), Instituto de Biociências, Rio Claropt

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