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

• Published in: PloS one 2016-03, Vol.11 (3), p.e0150764-e0150764
• Main Authors: Kaur, Preet Kamal, Tripathi, Neha, Desale, Jayesh, Neelagiri, Soumya, Yadav, Shailendra, Bharatam, Prasad V, Singh, Sushma
• Format: Article
• Language: English
• Subjects: Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Amino acids; Analysis; Arabinose; Atomic structure; Binding; Biology and Life Sciences; Biotechnology; Catalysis; Clostridium difficile; Clostridium thermocellum; Conserved Sequence; D-Ribose; Data processing; Drug development; E coli; Enzymatic activity; Enzyme activity; Enzymes; Fluorescence; Health education; Kinetics; Leishmania donovani; Leishmania donovani - genetics; Leishmania donovani - metabolism; Leishmaniasis; Models, Molecular; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis; Mutants; Mutation; Mycobacterium tuberculosis; Parasites; Parasitic diseases; Pharmaceutical sciences; Phosphates; Physical Sciences; Protein Binding; Protein Conformation; Protein structure; Proteins; Ribose; Ribose-5-phosphate isomerase; Risk factors; Sequence Alignment; Spectral analysis; Stability; Structural analysis; Structure-Activity Relationship; Substrate Specificity; Substrates; Trypanosoma cruzi; Tuberculosis; Vector-borne diseases; Visceral leishmaniasis
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0150764

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.