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

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...

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Autores principales: Morales-Luna, Laura, Hernández-Ochoa, Beatriz, Ramírez-Nava, Edson Jiovany, 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 María, Sierra-Palacios, Edgar, Castillo-Rodríguez, Rosa Angélica, Vega-García, Vanesa, Rufino-González, Yadira, Marcial-Quino, Jaime, Gómez-Manzo, Saúl
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402283/
https://www.ncbi.nlm.nih.gov/pubmed/32650494
http://dx.doi.org/10.3390/ijms21144831
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author Morales-Luna, Laura
Hernández-Ochoa, Beatriz
Ramírez-Nava, Edson Jiovany
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 María
Sierra-Palacios, Edgar
Castillo-Rodríguez, Rosa Angélica
Vega-García, Vanesa
Rufino-González, Yadira
Marcial-Quino, Jaime
Gómez-Manzo, Saúl
author_facet Morales-Luna, Laura
Hernández-Ochoa, Beatriz
Ramírez-Nava, Edson Jiovany
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 María
Sierra-Palacios, Edgar
Castillo-Rodríguez, Rosa Angélica
Vega-García, Vanesa
Rufino-González, Yadira
Marcial-Quino, Jaime
Gómez-Manzo, Saúl
author_sort Morales-Luna, Laura
collection PubMed
description 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 (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.
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spelling pubmed-74022832020-08-11 Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP(+) Molecule on Enzyme Stability Morales-Luna, Laura Hernández-Ochoa, Beatriz Ramírez-Nava, Edson Jiovany 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 María Sierra-Palacios, Edgar Castillo-Rodríguez, Rosa Angélica Vega-García, Vanesa Rufino-González, Yadira Marcial-Quino, Jaime Gómez-Manzo, Saúl Int J Mol Sci Article 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 (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. MDPI 2020-07-08 /pmc/articles/PMC7402283/ /pubmed/32650494 http://dx.doi.org/10.3390/ijms21144831 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Morales-Luna, Laura
Hernández-Ochoa, Beatriz
Ramírez-Nava, Edson Jiovany
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 María
Sierra-Palacios, Edgar
Castillo-Rodríguez, Rosa Angélica
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
title Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP(+) Molecule on Enzyme Stability
title_full Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP(+) Molecule on Enzyme Stability
title_fullStr Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP(+) Molecule on Enzyme Stability
title_full_unstemmed Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP(+) Molecule on Enzyme Stability
title_short Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP(+) Molecule on Enzyme Stability
title_sort characterizing the fused tvg6pd::6pgl protein from the protozoan trichomonas vaginalis, and effects of the nadp(+) molecule on enzyme stability
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402283/
https://www.ncbi.nlm.nih.gov/pubmed/32650494
http://dx.doi.org/10.3390/ijms21144831
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