TY - JOUR
T1 - Characterizing the fused tvg6pd::6pgl protein from the protozoan trichomonas vaginalis, and effects of the nadp+ molecule on enzyme stability
AU - Morales-Luna, Laura
AU - Hernández-Ochoa, Beatriz
AU - Ramírez-Nava, Edson Jiovany
AU - Martínez-Rosas, Víctor
AU - Ortiz-Ramírez, Paulina
AU - Fernández-Rosario, Fabiola
AU - González-Valdez, Abigail
AU - Cárdenas-Rodríguez, Noemí
AU - Serrano-Posada, Hugo
AU - Centeno-Leija, Sara
AU - Arreguin-Espinosa, Roberto
AU - Cuevas-Cruz, Miguel
AU - Ortega-Cuellar, Daniel
AU - de la Cruz, Verónica Pérez
AU - Rocha-Ramírez, Luz María
AU - Sierra-Palacios, Edgar
AU - Castillo-Rodríguez, Rosa Angélica
AU - Vega-García, Vanesa
AU - Rufino-González, Yadira
AU - Marcial-Quino, Jaime
AU - Gómez-Manzo, Saúl
N1 - Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2020/7/2
Y1 - 2020/7/2
N2 - 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.
AB - 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.
KW - 3D-structure
KW - Biochemical characterization
KW - G6PD
KW - Heterologous expression
KW - Trichomonas vaginalis
UR - http://www.scopus.com/inward/record.url?scp=85088169394&partnerID=8YFLogxK
U2 - 10.3390/ijms21144831
DO - 10.3390/ijms21144831
M3 - Artículo
C2 - 32650494
AN - SCOPUS:85088169394
SN - 1661-6596
VL - 21
SP - 1
EP - 24
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 14
M1 - 4831
ER -