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241.
Fernanda Ely José ES Nunes Evelyn K Schroeder Jeverson Frazzon Mário S Palma Diógenes S Santos Luiz A Basso 《BMC biochemistry》2008,9(1):13
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 (Mt CS), 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 Mt CS. The bifunctionality of Mt CS 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 Mt CS. 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 Mt CS and should thus pave the way for the rational design of antitubercular agents.242.
Density functional theory and quantum mechanics/molecular mechanics computations predict cob(I)alamin (Co(+)Cbx), a universal B(12) intermediate state, to be a pentacoordinated square pyramidal complex, which is different from the most widely accepted viewpoint of its tetracoordinated square planar geometry. The square pyramidality of Co(+)Cbx is inspired by the fact that a Co(+) ion, which has a dominant d(8) electronic configuration, forms a distinctive Co(+)--H interaction because of the availability of appropriately oriented filled d orbitals. This uniquely H-bonded Co(+)Cbx may have catalytic relevance in the context of thermodynamically uphill Co(2+)/Co(+) reduction that constitutes an essential component in a large variety of methyltransferases. 相似文献
243.
Z. Klusek W. Kozlowski Z. Waqar S. Datta I.V. Makarenko E.V. Rutkov A.N. Titkov 《Applied Surface Science》2005,252(5):1221-1227
Scanning tunnelling microscopy and current imaging tunnelling spectroscopy were used to observe electronic structure of the edges of monolayer graphite film deposited on the Ir(1 1 1) surface. The electronic structure derived from the tunnelling spectra revealed peak in electron local density of states very close to the Fermi level. This electronic state was interpreted in terms of localised edge state caused by the topology of the π electrons networks typical for the zig-zag edges. The observed maximum of local density of states at about 0.2 eV above the Fermi level was ascribed to the presence of resonant state caused by the appearance of disclinations centres in the vicinity of the graphite edges. 相似文献