Essential biochemical, biophysical and computational inputs on efficient functioning of Mycobacterium tuberculosis...

Mycobacterium tuberculosis (M. tuberculosis H₃₇Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include V_max and K_m for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. k_cat and catalytic efficiency of the reaction were 0.012±0.003 s^-1 and 0.00047±0.0001 μM/s^-1 respectively. These values were affected upon changing the standard conditions. Cations (Mg²⁺ and Mn²⁺) play important role in FtsY enzymatic activity as increasing Mg²⁺ decrease the activity. Mn²⁺on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.