Mutational and Structural Analysis of Conserved Residues in Ribose-5-Phosphate Isomerase B from Leishmania donovani: Role in Substrate Recognition and Conformational Stability.

Ribose-5-phosphate isomerase B from Leishmania donovani (LdRpiB) is one of the potential drug targets against visceral leishmaniasis. In the present study, we have targeted several conserved amino acids for mutational analysis (i.e. Cys69, His11, His102, His138, Asp45, Tyr46, Pro47 and Glu149) to gain crucial insights into their role in substrate binding, catalysis and conformational stability of the enzyme. All the eight LdRpiB variants were cloned, sequenced, expressed and purified. C69S, H102N, D45N and E149A mutants exhibited complete loss of enzyme activity indicating that they are indispensable for the enzyme activity. Kinetic parameters were altered in case of H138N, H11N and P47A variants; however Y46F exhibited similar kinetic behaviour as wild type. All the mutants except H138N exhibited altered protein structure as determined by CD and fluorescence spectral analysis. This data was supported by the atomic level details of the conformational changes and substrate binding using molecular dynamic simulations. LdRpiB also exhibited activity with D-form of various aldose substrates in the order of D-ribose > D-talose > D-allose > D-arabinose. Our study provides insights for better understanding of substrate enzyme interactions which can rationalize the process of drug design against parasite RpiB

Kinetic parameters for wild type <i>Ld</i>RpiB and its mutants.

<p>Kinetic parameters for wild type <i>Ld</i>RpiB and its mutants.</p

A graph plot of percentage relative fold change in fluorescence intensity of <i>Ld</i>RpiB wild type and its mutants.

<p>Data represents the mean ± S.D. from three experiments performed in duplicates, * represents <i>p</i> ≤ 0.05 and ** represents <i>p</i> ≤ 0.01.</p

Biophysical analysis of <i>Ld</i>RpiB wild type (wt) and various mutants.

<p>(A) CD spectra of <i>Ld</i>RpiB and its mutants from 190–250 nm. (B) Changes in percentage mean residue ellipticity of <i>Ld</i>RpiB and its mutants. Data represents the mean ± S.D. from three experiments performed in duplicates, * represents <i>p</i> ≤ 0.05, ** represents <i>p</i> ≤ 0.01 and *** represents <i>p</i> ≤ 0.001. (C) Fluorescence Emission spectra at 290 nm wavelength. (D) and (E) Changes observed in the emission maxima and fluorescence intensity of wild type RpiB and its mutants respectively. Data represents the mean ± S.D. from three experiments performed in duplicates, * represents <i>p</i> ≤ 0.05, ** represents <i>p</i> ≤ 0.01 and *** represents <i>p</i> ≤ 0.001.</p

Biochemical analysis of <i>Ld</i>RpiB wild type (wt) and various mutants.

<p>(A) Percentage relative enzyme activity. Kinetic parameters of <i>Ld</i>RpiB wild type (wt) and various mutants. (B) A graph plot of <i>K</i>_m values for wild type <i>Ld</i>RpiB and its mutants. (C) A graph plot of <i>k</i>_cat values for <i>Ld</i>RpiB wt and its mutants and (D) A graph plot of <i>k</i>_cat /<i>K</i>_m values for <i>Ld</i>RpiB wt and its mutants. Data represents the mean ± S.D. from three experiments performed in duplicates, * represents <i>p</i> ≤ 0.05, ** represents <i>p</i> ≤ 0.01 and *** represents <i>p</i> ≤ 0.001.</p

Sequence alignment of <i>Ld</i>RpiB amino acid sequence with amino acid sequence of <i>T</i>. <i>cruzi</i> RpiB and <i>H</i>. <i>sapiens</i> RpiA using Clustal W.

<p>Black dot represents the amino acids targeted for site directed mutagenesis, black box represents the amino acids present in the substrate binding pocket of <i>T</i>. <i>cruzi</i> and red box represents the other conserved amino acids.</p

Surface electrostatic potential of <i>Ld</i>RpiB.

<p>The residues shown in inset are responsible for interaction with the substrate PO₄^2- moiety.</p

Homology model validation parameters for template, <i>Ld</i>RpiB wild type and various mutants.

<p>Homology model validation parameters for template, <i>Ld</i>RpiB wild type and various mutants.</p

Substrate specificity of <i>Ld</i>RpiB using D- form of aldose sugars.

<p>(A) R5P, (B) D- Ribose, (C) D- Arabinose, (D) D- Talose and (E) D- Allose. Data represents the mean ± S.D. from three experiments performed in duplicates, * represents <i>p</i> ≤ 0.05, ** represents <i>p</i> ≤ 0.01 and *** represents <i>p</i> ≤ 0.001.</p

Residue decomposition analysis (GBSA) of residues (for C-form of R5P binding) showing differences among various mutants.

<p>The corresponding data for the F-form conformation is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150764#pone.0150764.s007" target="_blank">S7 Fig</a> (supporting information).</p