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

Abstract

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.

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

“…The UV-CD spectra did not show changes in the secondary structure of the protein (α-helices and β-sheets), when the protein was incubated with different NADP + concentrations, indicating that the NADP + molecule did not alter the folding of the protein at the level of the secondary structure and that the protective effect observed in the thermal inactivation and thermal stability trials could be due to the presence of the structural NADP + binding site in the TvG6PD::6PGL protein. This result is in agreement with that previously reported for the fused G6PD::6PGL from G. lamblia [45], where no alterations in molar ellipticity, either in the absence or presence of the NADP + molecule, were observed. This means that the shift in thermal stability and the thermal stability observed for the TvG6PD::6PGL protein in the presence of the NADP + molecule are due to a protective effect, as also reported for the human G6PD [44] and fused G6PD::6PGL from G. lamblia [45].…”

“…This result is in agreement with that previously reported for the fused G6PD::6PGL from G. lamblia [45], where no alterations in molar ellipticity, either in the absence or presence of the NADP + molecule, were observed. This means that the shift in thermal stability and the thermal stability observed for the TvG6PD::6PGL protein in the presence of the NADP + molecule are due to a protective effect, as also reported for the human G6PD [44] and fused G6PD::6PGL from G. lamblia [45].…”

“…When the TvG6PD::6PGL protein was incubated with 27 µM of NADP + , a slight protective effect in its resistance to protease digestion was observed. This result is in concordance with that previously observed for the fused G6PD::6PGL protein from G. lamblia [45], and human G6PD where a protective effect was observed when the protein was incubated with NADP + and the proteins became more resistant to protease digestion compared to without NADP + [45,47]. However, the TvG6PD::6PGL protein was more susceptible to trypsin protease degradation compared to the fused G6PD::6PGL protein from G. lamblia and the G6PD proteins from G. diazotrophicus and H. sapiens, where 0.4 mg/mL of trypsin was necessary to abolish 50% of the initial activity [23,43,45].…”

“…This result is in concordance with that previously observed for the fused G6PD::6PGL protein from G. lamblia [45], and human G6PD where a protective effect was observed when the protein was incubated with NADP + and the proteins became more resistant to protease digestion compared to without NADP + [45,47]. However, the TvG6PD::6PGL protein was more susceptible to trypsin protease degradation compared to the fused G6PD::6PGL protein from G. lamblia and the G6PD proteins from G. diazotrophicus and H. sapiens, where 0.4 mg/mL of trypsin was necessary to abolish 50% of the initial activity [23,43,45]. The TvG6PD::6PGL protein was thus more susceptible to trypsin digestion than the fused G6PD::6PGL protein from G. lamblia and human G6PD because the TvG6PD::6PGL protein features eight types of cleavage-specific trypsin digestion, as shown in Figure 7C, while the fused G6PD::6PGL from G. lamblia and human G6PD proteins contains only two specific trypsin cut sites ( Figure 7D).…”

“…However, when the TvG6PD::6PGL protein was incubated with a higher concentration of NADP + (500 µM), its residual activity was increased by 6 • C (a T 50 of 51.93 • C) without the NADP + molecule. This result is similar to that for the fused G6PD::6PGL protein from G. lamblia, whose T 50 increased by 6 • C (from 49.3 to 57.4 • C) when it was incubated in the presence of 500 µM of NADP + [45]. These results indicate that the protein became more thermoresistant than without NADP + , showing a shift of 6 • C in the presence of NADP + .…”