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Morales-Luna, Laura; Hernández-Ochoa, Beatriz; Ramírez-Nava, Edson; Martínez-Rosas, Víctor; Ortiz-Ramírez, Paulina; Fernández-Rosario, Fabiola; González-Valdez, Abigail; Cárdenas-Rodríguez, Noemí; Serrano-Posada, Hugo; Centeno-Leija, Sara; Arreguin-Espinosa, Roberto; Cuevas-Cruz, Miguel; Ortega-Cuellar, Daniel; Pérez de la Cruz, Verónica; Rocha-Ramírez, Luz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa; Vega-García, Vanesa; Rufino-González, Yadira; Marcial-Quino, Jaime; Gómez-Manzo, Saúl

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

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  • Media type: E-Article
  • Title: Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability
  • Contributor: Morales-Luna, Laura; Hernández-Ochoa, Beatriz; Ramírez-Nava, Edson; Martínez-Rosas, Víctor; Ortiz-Ramírez, Paulina; Fernández-Rosario, Fabiola; González-Valdez, Abigail; Cárdenas-Rodríguez, Noemí; Serrano-Posada, Hugo; Centeno-Leija, Sara; Arreguin-Espinosa, Roberto; Cuevas-Cruz, Miguel; Ortega-Cuellar, Daniel; Pérez de la Cruz, Verónica; Rocha-Ramírez, Luz; Sierra-Palacios, Edgar; Castillo-Rodríguez, Rosa; Vega-García, Vanesa; Rufino-González, Yadira; Marcial-Quino, Jaime; Gómez-Manzo, Saúl
  • imprint: MDPI AG, 2020
  • Published in: International Journal of Molecular Sciences
  • Language: English
  • DOI: 10.3390/ijms21144831
  • ISSN: 1422-0067
  • Keywords: Inorganic Chemistry ; Organic Chemistry ; Physical and Theoretical Chemistry ; Computer Science Applications ; Spectroscopy ; Molecular Biology ; General Medicine ; Catalysis
  • Origination:
  • Footnote:
  • Description: <jats:p>This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.</jats:p>
  • Access State: Open Access