Comprehensive kinetic characterization and inhibitor study of Ribonuclease E, a key enzyme of RNA metabolism from...

Ribonuclease E (RNase E) in Mycobacterium species is an essential gene that plays a crucial role in RNA metabolism. This is the first detailed kinetic characterization of recombinant RNase E and its deletion mutants, along with virtual high-throughput ligand screening (vHTS) and in vitro inhibitor studies from any Mycobacterium species. Recombinant wild-type RNase E and its deletion mutants of Mycobacterium smegmatis (M. smegmatis) were cloned, expressed and purified using Ni⁺²-NTA affinity chromatography. The optimal expression of recombinant His-RNase E was fine-tuned by adjusting various parameters, that included choice of Codon Plus(DE3) as expression host strain at 33 °C temperature and 0.1 mM IPTG concentration. Semi-quantitative RNase E activity was analyzed by an agarose gel-based assay using M. smegmatis total RNA as native substrate, depicting time-dependent degradation by the purified recombinant proteins. Kinetic characterization of wild-type and RNase E deletion mutants was performed by a liquid assay using methylene blue-RNA intercalation by monitoring the change in absorbance at 690 nm. The optimized enzyme concentration of 0.4 μg ml^-1 was utilized throughout the assay and the wild-type enzyme depicted maximum activity at 50 °C temperature and pH 7. The wild-type RNase E activity increased by 40 % and 8 % in presence of 5 mM Mn⁺² and Mg⁺² cations respectively. Maximum velocity (V_max) of the wild-type was 11.89 ± 0.1057 μg ml^-1 min^-1 and K_m was 28.26 ± 1.828 μg ml^-1, comparable with the N-terminal deletion mutant P-3 while the C-terminal deletion mutant P-2 demonstrates ∼3 folds lower V_max and ∼ 3 folds higher K_m as compared to the wild-type RNase E P-1. Being an essential gene, principle regulator of RNA metabolism and a promising antibacterial drug target, in silico vHTS of its highly conserved RNase domain and Molecular Dynamics (MD) simulations identified Sirolimus and levothyroxine as two top hit inhibitors. In vitro RNA degradation assay further confirmed Sirolimus as a non-competitive inhibitor of RNase E with K_i of 1.0 μM. In future, Sirolimus can be developed as potent anti-mycobacterial drug targetting RNase E after further modifications/ derivatization studies.