Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability
Year: 2020
Type: article
Abstract: 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.
DOI: 10.3390/ijms21144831
Authors: Laura Morales-Luna, Beatriz Hernández-Ochoa, Edson Ramírez-Nava, Víctor Martínez-Rosas, Paulina Ortiz-Ramírez, Fabiola Fernández-Rosario, Abigail González-Valdez, Noemí Cárdenas-Rodríguez, Hugo Serrano-Posada, Sara Centeno-Leija, Roberto Arreguin-Espinosa, Miguel Cuevas-Cruz, Daniel Ortega-Cuellar, Verónica Pérez de la Cruz, Luz Rocha-Ramírez, Edgar Sierra-Palacios, Rosa Castillo-Rodríguez, Vanesa Vega-García, Yadira Rufino-González, Jaime Marcial-Quino, Saúl Gómez-Manzo
Institutions: Secretaria de Salud, Instituto Nacional de Pediatria, Universidad Nacional Autónoma de México, Hospital Infantil de México Federico Gómez, Instituto Politécnico Nacional, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Secretaría de Ciencia, Humanidades, Tecnología e Innovación, Universidad de Colima, Instituto Nacional de Neurología y Neurocirugía, Universidad Autónoma de la Ciudad de México
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,112
Funder: Instituto Nacional de Pediatria
OpenAlex ID: W3041490220