PMID- 35036723 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20220119 IS - 2470-1343 (Electronic) IS - 2470-1343 (Linking) VI - 7 IP - 1 DP - 2022 Jan 11 TI - Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani. PG - 548-564 LID - 10.1021/acsomega.1c04967 [doi] AB - Pentose phosphate pathway (PPP) plays a crucial role in the maintenance of NADPH/NADP(+) homeostasis and provides protection against oxidative stress through detoxification of the reactive oxygen species. Ribulose-5-phosphate epimerase (RPE) participates in catalysis of the interconversion of ribulose-5-phosphate (Ru5P) to xylulose-5-phosphate (Xu5P) during PPP, however the structural attributes of this enzyme are still underexplored in many human pathogens including leishmanial parasites. The present study focuses upon cloning, purification and characterization of RPE of Leishmania donovani (LdRPE) using various biophysical and structural approaches. Sequence analysis has shown the presence of trypanosomatid-specific insertions at the N-terminus that are absent in humans and other eukaryotes. Gel filtration chromatography indicated recombinant LdRPE to exist as a dimer in the solution. Circular dichroism studies revealed a higher alpha helical content at physiological pH and temperature that comparatively varies with changing these parameters. Additionally, intrinsic fluorescence and quenching studies of LdRPE have depicted that tryptophan residues are mainly buried in the hydrophobic regions, and the recombinant enzyme is moderately tolerant to urea. Moreover, homology modeling was employed to generate the three-dimensional structure of LdRPE followed by molecular docking with the substrate, product, and substrate analogues. The modeled structure of LdRPE unravelled the presence of conserved active site residues as well as a single binding pocket for the substrate and product, while an in silico study suggested binding of substrate analogues into a similar pocket with more affinity than the substrate. Additionally, molecular dynamics simulation analysis has deciphered complexes of LdRPE with most of the ligands exhibiting more stability than its apo form and lesser fluctuations in active site residues in the presence of ligands. Altogether, our study presents structural insights into leishmanial RPE that could provide the basis for its implication to develop potent antileishmanials. CI - (c) 2021 The Authors. Published by American Chemical Society. FAU - Narsimulu, Bandigi AU - Narsimulu B AD - Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Hyderabad 500046, India. FAU - Qureshi, Rahila AU - Qureshi R AD - Private Location, Hyderabad 500019, India. FAU - Jakkula, Pranay AU - Jakkula P AD - Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Hyderabad 500046, India. FAU - Are, Sayanna AU - Are S AD - Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Hyderabad 500046, India. FAU - Qureshi, Insaf Ahmed AU - Qureshi IA AUID- ORCID: 0000-0001-7720-7067 AD - Department of Biotechnology & Bioinformatics, School of Life Sciences, University of Hyderabad, Prof. C. R. Rao Road, Hyderabad 500046, India. LA - eng PT - Journal Article DEP - 20211217 PL - United States TA - ACS Omega JT - ACS omega JID - 101691658 PMC - PMC8756792 COIS- The authors declare no competing financial interest. EDAT- 2022/01/18 06:00 MHDA- 2022/01/18 06:01 PMCR- 2021/12/17 CRDT- 2022/01/17 06:09 PHST- 2021/09/08 00:00 [received] PHST- 2021/11/25 00:00 [accepted] PHST- 2022/01/17 06:09 [entrez] PHST- 2022/01/18 06:00 [pubmed] PHST- 2022/01/18 06:01 [medline] PHST- 2021/12/17 00:00 [pmc-release] AID - 10.1021/acsomega.1c04967 [doi] PST - epublish SO - ACS Omega. 2021 Dec 17;7(1):548-564. doi: 10.1021/acsomega.1c04967. eCollection 2022 Jan 11.