The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase
| العنوان: | The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase |
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| المؤلفون: | José Eduardo Sacconi Nunes, Mario Sergio Palma, Luiz Augusto Basso, Evelyn Koeche Schroeder, Jeverson Frazzon, Fernanda Ely, Diógenes Santiago Santos |
| المساهمون: | Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Universidade Federal do Rio Grande do Sul (UFRGS), Universidade Estadual Paulista (Unesp) |
| المصدر: | BMC Biochemistry, Vol 9, Iss 1, p 13 (2008) Scopus Repositório Institucional da UNESP Universidade Estadual Paulista (UNESP) instacron:UNESP BMC Biochemistry Repositório Institucional da UFRGS Universidade Federal do Rio Grande do Sul (UFRGS) instacron:UFRGS |
| بيانات النشر: | Springer Nature |
| مصطلحات موضوعية: | molecular cloning, gene amplification, Flavin Mononucleotide, lyase, Chorismic Acid, lcsh:Animal biochemistry, Flavin mononucleotide, reduced nicotinamide adenine dinucleotide dehydrogenase, Biochemistry, chorismate synthase, chemistry.chemical_compound, synthetase, protein purification, Chorismic acid, Shikimate pathway, genetics, lcsh:QD415-436, shikimic acid, Bacteria (microorganisms), mass spectrometry, biology, flavine mononucleotide, Phosphorus-Oxygen Lyases, Oxidation-Reduction, Research Article, gel filtration chromatography, Chorismate synthase, enzymology, DNA sequence, flavine mononucleotide reductase, Mycobactin, chemistry, Catalysis, lcsh:Biochemistry, multidrug resistance, FMN reductase, spectrophotometry, enzyme mechanism, Enzyme kinetics, nicotinamide adenine dinucleotide, protein expression, lcsh:QP501-801, Molecular Biology, enzyme analysis, nonhuman, solvent effect, Base Sequence, spectrofluorometry, protein subunit, molecular weight, nucleotide sequence, Mycobacterium tuberculosis, Shikimic acid, NAD, bacterial DNA, Protein Subunits, biology.protein, biosynthesis, metabolism, oxidation reduction reaction |
| الوصف: | Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:23:33Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:30:05Z : No. of bitstreams: 1 2-s2.0-43749101877.pdf: 591246 bytes, checksum: 1eee79cbfc287d7cabedf0c0ed0b9136 (MD5) Made available in DSpace on 2014-05-27T11:23:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-05-22 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 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. Centro de Pesquisas Em Biologia Molecular e Funcional Pontifícia Universidade Católica Do Rio Grande Do Sul, RS 90619-900, Porto Alegre Instituto de Ciéncia e Tecnologia de Alimentos Universidade Federal Do Rio Grande Do Sul, RS 91501-970, Porto Alegre Departamento de Biologia/CEIS Universidade Estadual Paulista, SP 13506-900, Rio Claro Departamento de Biologia/CEIS Universidade Estadual Paulista, SP 13506-900, Rio Claro |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 1471-2091 |
| DOI: | 10.1186/1471-2091-9-13 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::39188596372b269e082ac101b9f05443Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....39188596372b269e082ac101b9f05443 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 14712091 |
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| DOI: | 10.1186/1471-2091-9-13 |