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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...
Autores principales: | , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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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. |
format | Online Article Text |
id | pubmed-7402283 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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