Biophysical and Structural Characterization of Ribulose-5-phosphate Epimerase from Leishmania donovani

Abstract

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.

Figure 1

Phylogenetic and sequence analysis of LdRPE. (A) Phylogenetic tree was generated using RPE protein sequences from various organisms through the neighbor-joining method. The numbers before the branch points delineate the confidence level of the relationship of the paired sequences determined by 1000 bootstrap statistical analysis. (B) Multiple sequence alignment of LdRPE was carried out with its counterpart from T. cruzi (Tcr), P. falciparum (Pfa), H. sapiens (Hsa), E. coli (Eco), and S. cerevisiae (Sce). The sequence is numbered according to LdRPE with conserved and similar residues highlighted as red and yellow, respectively. The RPE domain is presented as a box with green color, wherein insertions of amino acids are shown as blue boxes. Blue stars and red triangles indicate the substrate and product binding residues, respectively, with circled stars representing the residues binding with both the substrate and product. Green circles display the residues involved in dimeric interface formation, while pink square boxes denote the metal binding residues and red inverted triangles delineate the residue interacting with the substrate, product, and metal.

Figure 2

Purification and molecular weight determination of recombinant LdRPE. (A) 10% SDS-PAGE of purified LdRPE. Lane M shows the pre-stained protein marker, while lanes 1 and 2 indicate purified fractions. (B) Size exclusion chromatography profile of LdRPE displaying elution at 85.7 mL on a Superdex 16/600 200 pg corresponding to a molecular weight of 62.4 kDa. The inset demonstrates the resultant plot of protein standards such as ferritin (440 kDa), conalbumin (75 kDa), carbonic anhydrase (29.0 kDa), and RNase A (13.7 kDa) along with LdRPE (red circle) in buffer comprising 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5 and 100 mM KCl.

Figure 3

Effect of pH, temperature, and urea on the secondary structure of LdRPE. Far UV-CD spectra were taken from 250 to 195 nm using 5 μM of protein at pH 7.5 (A) and different pH (B). Normalized thermal denaturation plot with temperature from 20 to 90 °C (C). Far-UV CD spectra at 222 nm with various concentrations of urea were plotted and fit into a two-state equation (D).

Figure 4

Fluorescence emission spectra of LdRPE. Intrinsic fluorescence emission spectrum of LdRPE at pH 7.5 (A) and different pH (B). Extrinsic fluorescence spectra of LdRPE at various pHs with ANS (C). Fluorescence intensity and wavelength maxima as a function of pH (D). Effect of temperature on the emission spectra of apo (E) and complex of LdRPE with the substrate (G) and their respective thermal-induced denaturation curves (F,H).

Figure 5

Intrinsic fluorescence study of LdRPE with various divalent metal ions. Emission fluorescence spectra of LdRPE with increasing concentration (0.01–10 mM) of Mn²⁺ (A), Zn²⁺ (C), Fe²⁺ (E), and Mg²⁺ (G) and their respective modified Stern–Volmer plot to enumerate the binding constant of Mn²⁺ (B), Zn²⁺ (D), Fe²⁺ (F), and Mg²⁺ (H).

Figure 6

Intrinsic fluorescence quenching and unfolding studies of LdRPE. Fluorescence spectra of LdRPE with different concentrations (0–1 M) of acrylamide (A) and KI (C) and their respective Stern–Volmer plots enumerate the quenching constant (B,D). Effect of increasing urea concentration (0–8 M) on intrinsic fluorescence spectra of apo (E) and complex of LdRPE with the substrate (F) and their unfolding plots (G) fitted into a two-state equation.

Figure 7

Modeled three-dimensional structure of LdRPE. (A) Monomer of the 3D structure of LdRPE is represented in cartoon form, where α-helices, β-strands, and random coils are shown in red, yellow, and green colors, respectively. Zinc metal ions present in the active site are displayed as gray colored circles. (B) Interacting residues of the Zn²⁺ binding site are labeled and denoted as green sticks and metal ions as gray circles. (C) Dimeric structure of LdRPE contains two molecules shown in green and cyan colors with the inset depicting the interacting residues between two molecules labeled and represented as sticks, whereas black dashed lines indicate the intramolecular interactions.

Figure 8

Interaction studies of LdRPE with the substrate and product. Docked complexes of LdRPE with ligands were generated using SwissDock and represented as green ribbons. Subsequently, crystal structures of human RPE (cyan color) bound with the substrate (PDB ID: 3OVQ) and product (PDB ID: 3OVR) were superimposed onto the respective structures of LdRPE with Ru5P (A) and Xu5P (C) to compare the positions of the ligands. The residues of leishmanial and human RPEs interacting with Ru5P (B) and Xu5P (D) are labeled and displayed as green and cyan sticks, respectively.

Figure 9

Molecular docking of LdRPE with the substrate and its analogues. LdRPE was docked with the substrate and its analogues followed by their alignment to reveal the interacting residues. The residues of LdRPE interacting with Ru5P (A), Com A (B), Com B (C), Com C (D), Com D (E), and Com E (F) are presented as gray sticks and labeled.

Figure 10

MDS of apo LdRPE and its complexes. The plots display RMSD (A), R_g (B), and RMSF (C) for apo LdRPE and its docked complexes with Ru5P, Xu5P, Com A, Com B, Com C, Com D, and Com E.