Chemical- and thermal-induced unfolding of Leishmania donovani ribose-5-phosphate isomerase B: a single-tryptophan protein.

Abstract

Ribose-5-phosphate isomerase B (RpiB), a crucial enzyme of pentose phosphate pathway, was proposed to be a potential drug target for visceral leishmaniasis. In this study, we have analyzed the biophysical properties of Leishmania donovani RpiB (LdRpiB) enzyme to gain insight into its unfolding pathway under various chemical and thermal denaturation conditions by using fluorescence and CD spectroscopy. LdRpiB inactivation precedes the structural transition at lower concentrations of both urea and guanidine hydrochloride (GdHCl). 8-Anilinonapthalene 1-sulfonic (ANS) binding experiments revealed the presence of molten globule intermediate at 1.5 M GdHCl and a nonnative intermediate state at 6-M urea concentration. Acrylamide quenching experiments further validated the above findings, as solvent accessibility of tryptophan residues increased with increase in GdHCl and urea concentration. The recombinant LdRpiB was completely unfolded at 6 M GdHCl, whereas the enzyme molecule was resistant to complete unfolding even at 8-M urea concentration. The GdHCl- and urea-mediated unfolding involves a three-state transition process. Thermal-induced denaturation revealed complete loss of enzyme activity at 65 °C with only 20 % secondary structure loss. The formation of the well-ordered β-sheet structures of amyloid fibrils was observed after 55 °C which increased linearly till 85 °C as detected by thioflavin T dye. This study depicts the stability of the enzyme in the presence of chemical and thermal denaturants and stability-activity relationship of the enzyme. The presence of the intermediate states may have major implications in the way the enzyme binds to its natural ligand under various conditions. Also, the present study provides insights into the properties of intermediate entities of this important enzyme.

• Authors: Preet Kamal Kaur, Jakka S Supin, S Rashmi, Sushma Singh
• Journal: Applied biochemistry and biotechnology (Appl Biochem Biotechnol) Vol. 173 Issue 7 Pg. 1870-84 (Aug 2014) ISSN: 1559-0291 [Electronic] United States
• PMID: 24907042 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Recombinant Proteins
• Tryptophan
• Urea
• Aldose-Ketose Isomerases
• ribosephosphate isomerase
• Guanidine

Topics

• Aldose-Ketose Isomerases (chemistry, metabolism)
• Enzyme Stability (drug effects)
• Guanidine (pharmacology)
• Leishmania donovani (enzymology)
• Protein Unfolding (drug effects)
• Recombinant Proteins (chemistry, metabolism)
• Temperature
• Tryptophan
• Urea (pharmacology)