Crystal structure of leishmanial glucose-6-phosphate dehydrogenase and its implications for selective inhibitor...

Glucose-6-phosphate dehydrogenase (G6PDH) is a vital enzyme in trypanosomatid parasites, including Leishmania, where it generates NADPH to combat oxidative stress. In this study, Leishmania donovani G6PDH (LdG6PDH) was cloned, expressed, and purified to homogeneity using chromatographic methods. The purified enzyme existed as a dimer in solution, exhibited optimal activity near physiological pH, and displayed higher affinity for its cofactor (NADP) than for its substrate, glucose-6-phosphate (G6P). The enzymatic activity assay of leishmanial G6PDH revealed that ellagic acid (EA) and epigallocatechin gallate (EGCG) potently inhibit the enzyme, presenting a mixed-type mode of inhibition with respect to the substrate. Fluorescence spectroscopy established that most tryptophan residues are embedded within the hydrophobic core of LdG6PDH, while binding studies confirmed its physical interactions with ligands such as G6P, GLP, NADP, EA, and EGCG. Circular dichroism (CD) spectroscopy divulged a predominance of α-helical structure and substantiated the structural stability of the enzyme under different pH conditions. The crystal structure of LdG6PDH was resolved in both apo and ligand-bound forms, revealing three domains: a unique N-terminal domain, an NADP-binding domain, and a β + α domain. Notably, the ligand-bound structures demonstrated distinct binding positions for G6P and GLP compared to other known G6PDH structures. In silico studies identified lead compounds with a stronger affinity than NADP, indicating favorable physicochemical and pharmacokinetic properties, which were further validated through molecular dynamics simulations and MMPBSA analysis. Collectively, these findings elucidate the structural and functional properties of LdG6PDH, providing a foundation for the development of targeted inhibitors against leishmaniasis.