Leishmanial aspartyl-tRNA synthetase: Biochemical, biophysical and structural insights
| Autor: | Rahila Qureshi, Pranay Jakkula, Nooruddin Khan, Girish Ch. Panigrahi, Insaf A. Qureshi, K. Amith Kumar |
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| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Protein Conformation THP-1 Cells In silico Aspartate-tRNA Ligase Protozoan Proteins Aminoacylation 02 engineering and technology Nitric Oxide Biochemistry Cofactor Evolution Molecular 03 medical and health sciences chemistry.chemical_compound Mice Open Reading Frames Structural Biology Protein biosynthesis Animals Humans Enzyme kinetics Cloning Molecular Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences Cofactor binding Aspartic Acid biology Aminoacyl tRNA synthetase Temperature Tryptophan General Medicine Hydrogen-Ion Concentration 021001 nanoscience & nanotechnology Enzyme Pyrimidines RAW 264.7 Cells chemistry biology.protein 0210 nano-technology Leishmania donovani |
| Zdroj: | International journal of biological macromolecules. 165 |
| ISSN: | 1879-0003 |
| Popis: | Aminoacyl tRNA synthetases (aaRSs) are integral components of protein biosynthesis along with several non-canonical cellular processes. Inhibition studies of aaRSs presented these enzymes as promising drug targets in many pathogens, however aspartyl tRNA synthetase has not been studied in trypanosomatids despite its essentiality. Hence, full-length ORF of Leishmania donovani aspartyl tRNA synthetase (LdaspRS) was cloned and purified to homogeneity followed by molecular mass determination. The aminoacylation assay established that the purified protein performs its function optimally at physiological pH and temperature. The kinetic parameters of LdaspRS revealed the affinity of l -aspartate towards the enzyme to be very much lower than the cofactor. Our study also highlights the moonlighting function of LdaspRS to stimulate the pro-inflammatory cytokines and nitric oxide generation by host macrophage. Furthermore, CD and intrinsic tryptophan fluorescence measurements showed the changes in structural conformation at varying pH, denaturants and ligands. The modelled LdaspRS structure presented all the specific characteristics of class II aaRSs, while in silico study suggested binding of pyrimidine-derived inhibitors in its cofactor binding site with high affinity followed by validation using MD simulation. Altogether, this study could provide a platform for exploring LdaspRS to develop potential therapeutics against leishmaniasis. |
| Databáze: | OpenAIRE |
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