Abstract
Giardia lamblia is a single-celled eukaryotic parasite with a small genome and is considered an early divergent eukaryote. The pentose phosphate pathway (PPP) plays an essential role in the oxidative stress defense of the parasite and the production of ribose-5-phosphate. In this parasite, the glucose-6-phosphate dehydrogenase (G6PD) is fused with the 6-phosphogluconolactonase (6PGL) enzyme, generating the enzyme named G6PD::6PGL that catalyzes the first two steps of the PPP. Here, we report that the G6PD::6PGL is a bifunctional enzyme with two catalytically active sites. We performed the kinetic characterization of both domains in the fused G6PD::6PGL enzyme, as well as the individual cloned G6PD. The results suggest that the catalytic activity of G6PD and 6PGL domains in the G6PD::6PGL enzyme are more efficient than the individual proteins. Additionally, using enzymatic and mass spectrometry assays, we found that the final metabolites of the catalytic reaction of the G6PD::6PGL are 6-phosphoglucono-δ-lactone and 6-phosphogluconate. Finally, we propose the reaction mechanism in which the G6PD domain performs the catalysis, releasing 6-phosphoglucono-δ-lactone to the reaction medium. Then, this metabolite binds to the 6PGL domain catalyzing the hydrolysis reaction and generating 6-phosphogluconate. The structural difference between the G. lamblia fused enzyme G6PD::6PGL with the human G6PD indicate that the G6PD::6PGL is a potential drug target for the rational synthesis of novels anti-Giardia drugs.
Highlights
G. lamblia is a protist parasite causing giardiasis, a gastrointestinal disease
To corroborate if the fused enzyme glucose-6-phosphate dehydrogenase (G6PD)::6PGL of G. lamblia is a bifunctional enzyme, we determine the kinetic parameters of the G6PD and 6PGL domains of fused enzyme and the individual G6PD protein
When comparing the specificity constants for both enzymes in both glucose 6-phosphate (G6P) and NADP+ substrates, we found less efficiency for the individual G6PD enzyme for G6P and NADP+ concerning the fused enzyme, for G6P and NADP+ which again indicates that the G6PD enzyme must be folded together with the 6PGL domain to perform higher efficient catalysis, which is the feasible flow of the pathway
Summary
G. lamblia is a protist parasite causing giardiasis, a gastrointestinal disease. It is considered the most frequent parasitosis globally, and it is estimated that around 5 to 10% of the world population, which is approximately 300 million people, are affected by this microorganism [1,2,3]. PPP plays a crucial role in the parasite by generating the reduced nicotinamide-adenine-dinucleotide phosphate (NADPH). In the PPP oxidative phase, the first reaction involving the oxidation of glucose 6-phosphate (G6P) to (6PGL) is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). The third reaction is the decarboxylation of 6-phosphogluconate by the 6-phosphogluconate dehydrogenase (6PGDH); ribulose 5-phosphate and NADPH molecules are generated (Figure 1)
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