Identification of the NADP+ Structural Binding Site and Coenzyme Effect on the Fused G6PD:: 6PGL Protein from Giardia lamblia
Laura Morales-Luna, Abigail González-Valdez, Yudibeth Sixto-López, José Correa-Basurto, Beatriz Hernández-Ochoa, Noemí Cárdenas-Rodríguez, Rosa Angélica Castillo-Rodríguez, Daniel Ortega-Cuellar, Roberto Arreguin-Espinosa, Verónica Pérez de la Cruz, Hugo Serrano-Posada, Sara Centeno-Leija, Luz María Rocha-Ramírez, Edgar Sierra-Palacios, Alba Mónica Montiel-González, Yadira Rufino-González, Jaime Marcial-Quino, Saúl Gómez-Manzo
Giardia lambia is a flagellated protozoan parasite that lives in the small intestine and is the causal agent of giardiasis. It has been reported that G. lamblia exhibits glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway (PPP). Our group work demonstrated that the g6pd and 6pgl genes are present in the open frame that gives rise to the fused G6PD::6PGL protein; where the G6PD region is similar to the 3D structure of G6PD in Homo sapiens. The objective of the present work was to show the presence of the structural NADP+ binding site on the fused G6PD::6PGL protein and evaluate the effect of the NADP+ molecule on protein stability using biochemical and computational analysis. A protective effect was observed on the thermal inactivation, thermal stability, and trypsin digestions assays when the protein was incubated with NADP+. By molecular docking, we determined the possible structural-NADP+ binding site, which is located between the Rossmann fold of G6PD and 6PGL. Finally, molecular dynamic (MD) simulation was used to test the stability of this complex; it was determined that the presence of both NADP+ structural and cofactor increased the stability of the enzyme, which is in agreement with our experimental results.
Circular dichroism, Thermal stability, Protein folding, Secondary structure, Chemical stability, Biochemistry