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- Title
Gene Cloning, Recombinant Expression, Characterization, and Molecular Modeling of the Glycolytic Enzyme Triosephosphate Isomerase from Fusarium oxysporum.
- Authors
Hernández-Ochoa, Beatriz; Gómez-Manzo, Saúl; Alcaraz-Carmona, Erick; Serrano-Posada, Hugo; Centeno-Leija, Sara; Arreguin-Espinosa, Roberto; Cuevas-Cruz, Miguel; González-Valdez, Abigail; Mendoza-Espinoza, José Alberto; Acosta Ramos, Marcelo; Cortés-Maldonado, Leyda; Montiel-González, Alba Mónica; Pérez de la Cruz, Verónica; Rocha-Ramírez, Luz María; Marcial-Quino, Jaime; Sierra-Palacios, Edgar
- Abstract
Triosephosphate isomerase (TPI) is a glycolysis enzyme, which catalyzes the reversible isomerization between dihydroxyactetone-3-phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP). In pathogenic organisms, TPI is essential to obtain the energy used to survive and infect. Fusarium oxisporum (Fox) is a fungus of biotechnological importance due to its pathogenicity in different organisms, that is why the relevance of also biochemically analyzing its TPI, being the first report of its kind in a Fusarium. Moreover, the kinetic characteristics or structural determinants related to its function remain unknown. Here, the Tpi gene from F. oxysporum was isolated, cloned, and overexpressed. The recombinant protein named FoxTPI was purified (97% purity) showing a molecular mass of 27 kDa, with optimal activity at pH 8.0 and and temperature of 37 °C. The values obtained for Km and Vmax using the substrate GAP were 0.47 ± 0.1 mM, and 5331 μmol min−1 mg−1, respectively. Furthemore, a protein structural modeling showed that FoxTPI has the classical topology of TPIs conserved in other organisms, including the catalytic residues conserved in the active site (Lys12, His94 and Glu164). Finally, when FoxTPI was analyzed with inhibitors, it was found that one of them inhibits its activity, which gives us the perspective of future studies and its potential use against this pathogen.
- Subjects
TRIOSE-phosphate isomerase; MOLECULAR cloning; MOLECULAR models; RECOMBINANT proteins; MOLECULAR weights; FUSARIUM oxysporum
- Publication
Microorganisms, 2020, Vol 8, Issue 1, p40
- ISSN
2076-2607
- Publication type
Article
- DOI
10.3390/microorganisms8010040