Abstract
6-phosphogluconate dehydrogenase (6PGDH) participates in pentose phosphate pathway of glucose metabolism by catalyzing oxidative decarboxylation of 6-phsophogluconate (6PG) and its absence has been lethal for several eukaryotes. Despite being a validated drug target in many organisms like Plasmodium, the enzyme has not been explored in leishmanial parasites. In the present study, 6PGDH of Leishmania donovani (Ld6PGDH) is cloned and purified followed by its characterization using biochemical and structural approaches. Ld6PGDH lacks the glycine-serine-rich sequence at its C-terminal that is present in other eukaryotes including humans. Leishmanial 6PGDH possesses more affinity for substrate (6PG) and cofactor (NADP) in comparison to that of human. The enzymatic activity is inhibited by gentamicin and cefuroxime through competitive mode with functioning more potently towards leishmanial 6PGDH than its human counterpart. CD analysis has shown higher α-helical content in the secondary structure of Ld6PGDH, while fluorescence studies revealed that tryptophan residues are not completely accessible to solvent environment. The three-dimensional structure was generated through homology modelling and docked with substrate and cofactor. The docking studies demonstrated two separate binding pockets for 6PG and NADP with higher affinity for the cofactor binding, and Asn105 is interacting with substrate as well as the cofactor. Additionally, MD simulation has shown complexes of Ld6PGDH with 6PG and NADP to be more stable than its apo form. Altogether, the present study might provide the foundation to investigate this enzyme as potential target against leishmaniasis.
Key points
• Ld6PGDH enzymatic activity is competitively inhibited by gentamicin and cefuroxime.
• It displays more helical contents and all structural characteristics of 6PGDH family.
• Interaction studies demonstrate higher affinity of cofactor than substrate for Ld6PGDH.
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All data generated or analyzed during this study are included in this published article (and its supplementary information files).
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Acknowledgements
The authors are thankful to Dr. N.P. Prabhu for his suggestions during this work and Prof. Swati Saha for providing Leishmania donovani genomic DNA. We also acknowledge DST-FIST and UGC-SAP sponsored instruments facilities of Department of Biotechnology & Bioinformatics, School of Life Sciences as well as Centre for Modelling Simulation & Design (CMSD), University of Hyderabad, Hyderabad for computational resources for MD simulation studies. PJ and BN acknowledge ICMR and UGC for the research fellowships.
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This work was financially supported by a grant from Science and Engineering Research Board, India (Project No. EMR/2016/007746).
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PJ conducted experiments and analyzed the data with the help of BN. PJ wrote the draft of the manuscript. IAQ conceived and supervised the study along with editing the manuscript.
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Jakkula, P., Narsimulu, B. & Qureshi, I.A. Biochemical and structural insights into 6-phosphogluconate dehydrogenase from Leishmania donovani. Appl Microbiol Biotechnol 105, 5471–5489 (2021). https://doi.org/10.1007/s00253-021-11434-4
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DOI: https://doi.org/10.1007/s00253-021-11434-4