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Open AccessArticle

Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia

1
Laboratorio de Bioquímica Genética, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
2
Consejo Nacional de Ciencia y Tecnología (CONACYT), Laboratorio de Agrobiotecnología, Tecnoparque CLQ, Universidad de Colima, Carretera los Limones-Loma de Juárez, Colima 28629, Mexico
3
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad de Mexico 04510, Mexico
4
Laboratorio de Nutrición Experimental, Instituto Nacional de Pediatría, Secretaría de Salud 04530, Mexico
5
Laboratorio de Neurociencias, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
6
Laboratorio de Inmunoquímica, Hospital Infantil de México Federico Gómez, Ciudad de Mexico 06720, Mexico
7
Colegio de Ciencias y Humanidades, Plantel Casa Libertad, Universidad Autónoma de la Ciudad de México, Ciudad de México 09620, Mexico
8
Laboratorio de Parasitología Experimental, Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
9
CONACYT-Instituto Nacional de Pediatría, Secretaría de Salud, Ciudad de México 04530, Mexico
10
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, S.S.A., Ciudad de Mexico 14269, México
11
Unidad de Investigación en Biología Computacional y Diseño de Fármacos, Hospital Infantil de México Federico Gómez, Mexico City 06720, Mexico
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(9), 2518; https://doi.org/10.3390/ijms19092518
Received: 13 July 2018 / Revised: 18 August 2018 / Accepted: 22 August 2018 / Published: 25 August 2018
Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway and is highly relevant in the metabolism of Giardia lamblia. Previous reports suggested that the G6PD gene is fused with the 6-phosphogluconolactonase (6PGL) gene (6pgl). Therefore, in this work, we decided to characterize the fused G6PD-6PGL protein in Giardia lamblia. First, the gene of g6pd fused with the 6pgl gene (6gpd::6pgl) was isolated from trophozoites of Giardia lamblia and the corresponding G6PD::6PGL protein was overexpressed and purified in Escherichia coli. Then, we characterized the native oligomeric state of the G6PD::6PGL protein in solution and we found a catalytic dimer with an optimum pH of 8.75. Furthermore, we determined the steady-state kinetic parameters for the G6PD domain and measured the thermal stability of the protein in both the presence and absence of guanidine hydrochloride (Gdn-HCl) and observed that the G6PD::6PGL protein showed alterations in the stability, secondary structure, and tertiary structure in the presence of Gdn-HCl. Finally, computer modeling studies revealed unique structural and functional features, which clearly established the differences between G6PD::6PGL protein from G. lamblia and the human G6PD enzyme, proving that the model can be used for the design of new drugs with antigiardiasic activity. These results broaden the perspective for future studies of the function of the protein and its effect on the metabolism of this parasite as a potential pharmacological target. View Full-Text
Keywords: Giardia lamblia; G6PD; purification; recombinant expression; bioinformatics analysis; three-dimensional structure Giardia lamblia; G6PD; purification; recombinant expression; bioinformatics analysis; three-dimensional structure
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Morales-Luna, L.; Serrano-Posada, H.; González-Valdez, A.; Ortega-Cuellar, D.; Vanoye-Carlo, A.; Hernández-Ochoa, B.; Sierra-Palacios, E.; Rufino-González, Y.; Castillo-Rodríguez, R.A.; Pérez de la Cruz, V.; Moreno-Vargas, L.; Prada-Gracia, D.; Marcial-Quino, J.; Gómez-Manzo, S. Biochemical Characterization and Structural Modeling of Fused Glucose-6-Phosphate Dehydrogenase-Phosphogluconolactonase from Giardia lamblia. Int. J. Mol. Sci. 2018, 19, 2518.

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