دانلود رایگان مقاله خصوصیات بیوشیمیایی و بیوفیزیکی لیشمانیا دونووانی

Abstract

γ-glutamylcysteine synthetase (Gcs) is a vital enzyme catalyzing the first and rate limiting step in the trypanothione biosynthesis pathway, the ATP-dependent ligation of L-Glutamate and L-Cysteine to form gamma-glutamylcysteine. The Trypanothione biosynthesis pathway is unique metabolic pathway essential for trypanosomatid survival rendering Gcs as a potential drug target. Here we report the cloning, expression, purification and characterization of L. donovani Gcs. Three other constructs of Gcs (GcsN, GcsC and GcsT) were designed on the basis of S. cerevisiae and E. coli Gcs crystal structures. The study shows Gcs possesses ATPase activity even in the absence of substrates L-glutamate and L-Cysteine. Divalent ions however plays an indispensable role in LdGcs ATPase activity. Isothermal titration calorimetry and fluorescence studies illustrates that L. donovani Gcs binds substrate in order ATP 4L-glutamate4Lcysteine with Glu 92 and Arg 498 involved in ATP hydrolysis and Glu 92, Glu 55 and Arg 498 involved in glutamate binding. Homology modeling and molecular dynamic simulation studies provided the structural rationale of LdGcs catalytic activity and emphasized on the possibility of involvement of three Mg2þ ions along with Glutamates 52, 55, 92, 99, Met 322, Gln 328, Tyr 397, Lys 483, Arg 494 and Arg 498 in the catalytic function of L. donovani Gcs.

3.1.13. Molecular dynamic simulations

Substrate binding sites were obtained by extracting ligands from ScGcs crystal structures (PDB ID- 3IG8, 3IG5 and 3LVW). Molecular dynamic (MD) simulations and analysis were performed using GROMACS 4.6.5 simulation package [50] adopting CHARMM force field parameters [51]. LdGcs homology model with ADP, L-glutamate, L-cysteine and 3 Mg2þ ions was taken as starting point for simulation. The topology of ligands ADP, L-glutamate and L-cysteine were created using swissparam server [52]. The protein ligand complex was solvated into a cubic box of TIP3P water model. The complex was neutralized by using 18 Naþ ion. Energy minimization was done using steepest descent method and convergent crieteria of 10 KJ/Mol followed by dynamics simulations of the whole system in the NVT and NPT ensemble at 293 K temperature with a time step of 2 ps. The electrostatic interactions were calculated using the Particle Mesh Ewald summation method [53] while constraints were applied on all bonds using the LINCS [54] algorithm. The equilibrated system was subjected to final MD production run of 10 ns. Root mean square deviation (RMSD) of Cα residues and ligands and root mean square fluctuation (RMSF) of complex were calculated using g_rms and g_rmsf commands respectively. Graphs were generated using Grace Program. The various frames generated were visually analyzed by Chimera 1.6.1 [55].