Characterizing the Fused TvG6PD::6PGL Protein from the ProtozoanTrichomonas vaginalis, and Effects of the NADP(+)Molecule on Enzyme Stability
Por:
Morales-Luna, Laura, Hernandez-Ochoa, Beatriz, Jiovany Ramirez-Nava, Edson, Martinez-Rosas, Victor, Ortiz-Ramirez, Paulina, Fernandez-Rosario, Fabiola, Gonzalez-Valdez, Abigail, Cardenas-Rodriguez, Noemi, Serrano-Posada, Hugo, Centeno-Leija, Sara, Arreguin-Espinosa, Roberto, Cuevas-Cruz, Miguel, Ortega-Cuellar, Daniel, Perez de la Cruz, Veronica, Maria Rocha-Ramirez, Luz, Sierra-Palacios, Edgar, Angelica Castillo-Rodriguez, Rosa, Vega-Garcia, Vanesa, Rufino-Gonzalez, Yadira, Marcial-Quino, Jaime, Gomez-Manzo, Saul
Publicada:
1 jul 2020
Resumen:
This report describes a functional and structural analysis of fused
glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase
protein from the protozoanTrichomonas vaginalis(T. vaginalis). The
glucose-6-phosphate dehydrogenase (g6pd) gene fromT.vaginaliswas
isolated by PCR and the sequence of the product showed that is fused
with6pglgene. The fused Tvg6pd::6pglgene 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 (K-d) 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.
Filiaciones:
Morales-Luna, Laura:
Secretaria Salud, Lab Bioquim Genet, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Univ Nacl Autonoma Mexico, Ciencias Biol, Ciudad De Mexico 04510, Mexico
Hernandez-Ochoa, Beatriz:
Secretaria Salud, Lab Inmunoquim, Hosp Infantil Mexico Federico Gomez, Ciudad De Mexico 06720, Mexico
Inst Politecn Nacl, Escuela Nacl Ciencias Biol, Programa Posgrad Biomed & Biotecnol Mol, Ciudad De Mexico 11340, Mexico
Jiovany Ramirez-Nava, Edson:
Secretaria Salud, Lab Bioquim Genet, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Univ Nacl Autonoma Mexico, Ciencias Biol, Ciudad De Mexico 04510, Mexico
Martinez-Rosas, Victor:
Secretaria Salud, Lab Bioquim Genet, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Inst Politecn Nacl, Escuela Nacl Ciencias Biol, Programa Posgrad Biomed & Biotecnol Mol, Ciudad De Mexico 11340, Mexico
Ortiz-Ramirez, Paulina:
Secretaria Salud, Lab Bioquim Genet, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Fernandez-Rosario, Fabiola:
Secretaria Salud, Lab Bioquim Genet, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Gonzalez-Valdez, Abigail:
Univ Nacl Autonoma Mexico, Inst Invest Biomed, Dept Biol Mol & Biotecnol, Ciudad De Mexico 04510, Mexico
Cardenas-Rodriguez, Noemi:
Secretaria Salud, Lab Neurociencias, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Serrano-Posada, Hugo:
Univ Colima, Lab Agrobiotecnol, Consejo Nacl Ciencia & Tecnol CONACYT, Tecnoparque CLQ,Carretera Limones Loma Juarez, Colima 28629, Mexico
Centeno-Leija, Sara:
Univ Colima, Lab Agrobiotecnol, Consejo Nacl Ciencia & Tecnol CONACYT, Tecnoparque CLQ,Carretera Limones Loma Juarez, Colima 28629, Mexico
Arreguin-Espinosa, Roberto:
Univ Nacl Autonoma Mexico, Inst Quim, Dept Quim Biomacromol, Ciudad De Mexico 04510, Mexico
Cuevas-Cruz, Miguel:
Univ Nacl Autonoma Mexico, Inst Quim, Dept Quim Biomacromol, Ciudad De Mexico 04510, Mexico
Ortega-Cuellar, Daniel:
Secretaria Salud, Lab Nutr Expt, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Perez de la Cruz, Veronica:
Secretaria Salud, Dept Neuroquim, Inst Nacl Neurol & Neurocirugia Manuel Velasco Su, Ciudad De Mexico 14269, Mexico
Maria Rocha-Ramirez, Luz:
Hosp Infantil Mexico Dr Federico Gomez, Unidad Invest Enfermedades Infecciosas, Dr Marquez 162, Col Doctores 06720, Delegacion Cuau, Mexico
Sierra-Palacios, Edgar:
Univ Autonoma Ciudad Mexico, Plantel Casa Libertad, Colegio Ciencias & Humanidades, Ciudad De Mexico 09620, Mexico
Angelica Castillo-Rodriguez, Rosa:
Secretaria Salud, Inst Nacl Pediat, Consejo Nacl Ciencia & Tecnol CONACYT, Ciudad De Mexico 04530, Mexico
Vega-Garcia, Vanesa:
Univ Nacl Autonoma Mexico, Fac Ciencias, Ciudad De Mexico 04510, Mexico
Rufino-Gonzalez, Yadira:
Secretaria Salud, Lab Parasitol Expt, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
Marcial-Quino, Jaime:
Secretaria Salud, Inst Nacl Pediat, Consejo Nacl Ciencia & Tecnol CONACYT, Ciudad De Mexico 04530, Mexico
Gomez-Manzo, Saul:
Secretaria Salud, Lab Bioquim Genet, Inst Nacl Pediat, Ciudad De Mexico 04530, Mexico
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