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

Abstract

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.

Identification of the NADP+ Structural Binding Site and Coenzyme Effect on the Fused G6PD::6PGL Protein from Giardia lamblia

“…The G6PD::6PGL protein was expressed and purified as previously reported by Morales-Luna et al [19]. The E. coli BL21(DE3)∆zwf ::kan r cells containing the pET-3a/g6pd::6pgl vector were induced with 1 mM of isopropyl-β-d-thiogalactoside (IPTG) for 18 h at 25 • C in Luria Bertani (LB) culture medium.…”

“…The cells were pelleted by centrifugation, suspended in lysis buffer, and disrupted by sonication. Then, by centrifugation (10,000× g for 15 min at 4 • C), the crude extract was obtained and used for protein purification employing a 2 ,5 -ADP Sepharose 4B affinity column (GE Healthcare, Chicago, IL, USA) and a Sephacryl 200 (16/60) gel filtration column (GFC) (GE Healthcare, Chicago, IL, USA) [19]. Purified G6PD::6PGL protein was analyzed in 12% SDS-PAGE gel [20] and stained with colloidal Coomassie Brilliant Blue (R-250) (Sigma-Aldrich, San Luis, MO, USA).…”

“…On the other hand, for NADP + Gasteiger charges were assigned, considering that at physiological pH is charged (−3). The G6PD::6PGL model was obtained from a previous work [19] (Supplementary Materials g6pd.6pgl.pdb) and refined using the ModRefiner server [30] (Supplementary Materials g6pd_clean-rama.pdf). For G6PD::6PGL only polar hydrogens were considered, which are hydrogen atoms that are bonded to electronegative atoms (O, N, S) that are capable of forming hydrogen bonds; and charges were assigned Kollman united-atom library, which fits the electrostatic potential to points selected on a set of concentric spheres around each atom; the sum of all atomic charges is equal to that of the overall charge of the system [31] and the AD4.1_bound forcefield was employed in molecular docking [29].…”

“…Recent studies accomplished by Stover et al [18] and Morales-Luna et al [19] observed that the glucose-6-phosphate dehydrogenase (g6pd) and 6-phosphogluconolactonase (6pgl) genes were present in one open frame g6pd::6pgl gene, and that at the protein level it was possible to determine the existence of two functional domains present in the same (G6PD and 6PGL) sequenced regions. Furthermore, they observed that the amino acid sequence corresponding to the G6PD region on the fused G6PD::6PGL from G. lamblia was similar with the 3D structure of H. sapiens G6PD (PDB entry 2BH9).…”

“…Furthermore, they observed that the amino acid sequence corresponding to the G6PD region on the fused G6PD::6PGL from G. lamblia was similar with the 3D structure of H. sapiens G6PD (PDB entry 2BH9). On the other hand, the G6PD protein from G. lamblia, despite being fused with the 6PGL, maintains the same structural conformation as the G6PD of other organisms, since it is also a dimer made up of two symmetrically located subunits and each monomer binds to a molecule of catalytic NADP + coenzyme [19], in the Rossmann type folding domain. However, the existence of the structural NADP + binding site on the fused G6PD::6PGL protein from G. lamblia was not demonstrated.…”