TY - JOUR
T1 - Biochemical characterization and structural modeling of fused glucose-6-phosphate dehydrogenase-phosphogluconolactonase from Giardia lamblia
AU - Morales-Luna, Laura
AU - Serrano-Posada, Hugo
AU - González-Valdez, Abigail
AU - Ortega-Cuellar, Daniel
AU - Vanoye-Carlo, America
AU - Hernández-Ochoa, Beatriz
AU - Sierra-Palacios, Edgar
AU - Rufino-González, Yadira
AU - Castillo-Rodríguez, Rosa Angélica
AU - de la Cruz, Verónica Pérez
AU - Moreno-Vargas, Liliana
AU - Prada-Gracia, Diego
AU - Marcial-Quino, Jaime
AU - Gómez-Manzo, Saúl
N1 - Publisher Copyright:
© 2018 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2018/9
Y1 - 2018/9
N2 - 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.
AB - 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.
KW - Bioinformatics analysis
KW - G6PD
KW - Giardia lamblia
KW - Purification
KW - Recombinant expression
KW - Three-dimensional structure
UR - http://www.scopus.com/inward/record.url?scp=85052532316&partnerID=8YFLogxK
U2 - 10.3390/ijms19092518
DO - 10.3390/ijms19092518
M3 - Artículo
C2 - 30149622
AN - SCOPUS:85052532316
SN - 1661-6596
VL - 19
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 9
M1 - 2518
ER -