Harish Shukla

Distant Phe345 mutation compromises the stability and activity of Mycobacterium tuberculosis isocitrate lyase by modulating its structural flexibility

Isocitrate lyase (ICL), a potential anti-tubercular drug target, catalyzes the first step of the glyoxylate shunt. In the present investigation, we studied the conformational flexibility of MtbICL to better understand its stability and catalytic activity. Our biochemical results showed that a point mutation at Phe345, which is topologically distant (>10 Å) to the active site signature sequence (189KKCGH193), completely abolishes the activity of the enzyme. In depth computational analyses were carried out for understanding the structural alterations using molecular dynamics, time-dependent secondary structure and principal component analysis. The results showed that the mutated residue increased the structural flexibility and induced conformational changes near the active site (residues 170–210) and in the C-terminal lid region (residues 411–428). Both these regions are involved in the catalytic activity of MtbICL. Upon mutation, the residual mobility of the enzyme increased, resulting in a decrease in the stability, which was confirmed by the lower free energy of stabilization in the mutant enzyme suggesting the destabilization in the structure. Our results have both biological importance and chemical novelty. It reveals internal dynamics of the enzyme structure and also suggests that regions other than the active site should be exploited for targeting MtbICL inhibition and development of novel anti-tuberculosis compounds.

Structure-Based Screening And Molecular Dynamics Simulations Offer Novel Natural Compounds As Potential Inhibitors Of Mycobacterium Tuberculosis Isocitrate Lyase

Mycobacterium tuberculosis is the etiological agent of tuberculosis in humans and is responsible for more than two million deaths annually. M. tuberculosis isocitrate lyase (MtbICL) catalyzes the first step in the glyoxylate cycle, plays a pivotal role in the persistence of M. tuberculosis, which acts as a potential target for an anti-tubercular drug. To identify the potential anti-tuberculosis compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,748) against the MtbICL structure. The ligands were docked against MtbICL in three sequential docking modes that resulted in 340 ligands having better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 27 compounds were found to fit well with re-docking studies. After refinement by molecular docking and drug-likeness analyses, three potential inhibitors (ZINC1306071, ZINC2111081, and ZINC2134917) were identified. These three ligands and the reference compounds were further subjected to molecular dynamics simulation and binding energy analyses to compare the dynamic structure of protein after ligand binding and the stability of the MtbICL and bound complexes. The binding free energy analyses were calculated to validate and capture the intermolecular interactions. The results suggested that the three compounds had a negative binding energy with −96.462, −143.549, and −122.526 kJ mol−1 for compounds with IDs ZINC1306071, ZINC2111081, and ZINC2134917, respectively. These lead compounds displayed substantial pharmacological and structural properties to be drug candidates. We concluded that ZINC2111081 has a great potential to inhibit MtbICL and would add to the drug discovery process against tuberculosis.

A combined biochemical and computational studies of the rho-class glutathione s-transferase sll1545 of Synechocystis PCC 6803.

Peroxides are one of the most important radicals that cause oxidative stress. Certain Glutathione S-transferases (GSTs) have been reported to show peroxidase activity. We report a novel peroxidase activity of Synechocystis GST- sll1545. The recombinant protein was purified to homogeneity and characterized. Low Km (0.109μM) and high Vmax (0.663μmolmin-1) values suggest a high preference of sll1545 for cumenehydroperoxide. Disc inhibition assay confirmed the ability of the enzyme to protect cells against peroxide-induced damage. sll1545 has very low sequence and structural similarity with theta and alpha class GSTs that exhibit glutathione-dependent peroxidase activity. Recent data from our laboratory shows that sll1545 is also strongly active against dichloroacetate (DCA), which is a characteristic of zeta class GST. Interestingly, sll1545 shows less than 20% sequence identity with zeta class GST. Molecular dynamic simulation results show that sll1545 was much more structurally different from alpha/theta classes. Our results suggest that sll1545 shows structural variation from zeta, theta/alpha classes of GSTs but have related enzymatic activity. Phylogenetic analysis reveal that sll1545 is evolutionally very distinct from the known GSTs. Overall, the data suggest that Synechocystis sll1545 does not belong to any known GST class and represent a novel GST class, which we have named rho.

Alterations in conformational topology and interaction dynamics caused by L418A mutation leads to activity loss of Mycobacterium tuberculosis isocitrate lyase

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a key enzyme of the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate and is a potential antituberculosis drug target. The aim of this research was to explore the structural alterations induced by L418A point mutation that caused the loss of enzyme activity. In-depth structural analyses were carried out for understanding the influence of L418A mutation using techniques, viz. molecular dynamics, principal component analysis, time-dependent secondary structure, residue interaction network and molecular docking. Since L418A mutation site is structurally far from the active site, it cannot influence the binding of the substrate directly. Our results showed that collective motions, residual mobility, and flexibility of the enzyme increased upon mutation. The mutated residue changed the global conformational dynamics of the system along with the residue-residue interaction network, leading to a loss of the enzyme activity. The docking results suggest that L418A mutation influenced the binding interactions of the substrate with several residues in the active site of MtbICL. This study provides information on the structural dynamics of MtbICL and highlights the importance of residue level interactions in the protein. Thus, our results may provide significant guidance to the scientific community engaged in designing potent inhibitors targeting MtbICL.

Identification of potential inhibitors of Fasciola gigantica thioredoxin1: computational screening, molecular dynamics simulation, and binding free energy studies

Fasciola gigantica is the causative organism of fascioliasis and is responsible for major economic losses in livestock production globally. F. gigantica thioredoxin1 (FgTrx1) is an important redox-active enzyme involved in maintaining the redox homeostasis in the cell. To identify a potential anti-fasciolid compound, we conducted a structure-based virtual screening of natural compounds from the ZINC database (n = 1,67,740) against the FgTrx1 structure. The ligands were docked against FgTrx1 and 309 ligands were found to have better docking score. These compounds were evaluated for Lipinski and ADMET prediction, and 30 compounds were found to fit well for re-docking studies. After refinement by molecular docking and drug-likeness analysis, three potential inhibitors (ZINC15970091, ZINC9312362, and ZINC9312661) were identified. These three ligands were further subjected to molecular dynamics simulation (MDS) to compare the dynamics and stability of the protein structure after binding of the ligands. The binding free energy analyses were calculated to determine the intermolecular interactions. The results suggested that the two compounds had a binding free energy of –82.237, and –109.52 kJ.mol−1 for compounds with IDs ZINC9312362 and ZINC9312661, respectively. These predicted compounds displayed considerable pharmacological and structural properties to be drug candidates. We concluded that these two compounds could be potential drug candidates to fight against F. gigantica parasites.

UDP-N-Acetylglucosamine enolpyruvyl transferase (MurA) of Acinetobacter baumannii (AbMurA): Structural and functional properties.

Peptidoglycan (PG) is the key component of the bacterial cell wall. The enzyme UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridinediphospho-N-acetylglucosamine (UNAG), which is the first committed step of PG biosynthesis. Here, we present the biochemical and structural features of the MurA enzyme of the opportunistic pathogen Acinetobacter baumannii (AbMurA). The recombinant AbMurA exists as a monomer in solution and shows optimal activity at pH 7.5 and 37°C. The Km for UDP-N-acetylglucosamine was 1.062±0.09mM and for PEP was 1.806±0.23mM. The relative enzymatic activity was inhibited ∼3 fold in the presence of 50mM fosfomycin (FFQ). Superimposition of the AbMurA model with E. coli demonstrated key structural similarity in the FFQ-binding site. AbMurA also has a surface loop that contains the active site Cys116 that interact with FFQ. Sequence analysis indicates the presence of the five conserved amino acids, i.e., K22, C116, D306, D370 and L371, required for the functional activity like other MurA enzymes from different bacteria. MurA enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be a promising drug target.

Structural insights into natural compounds as inhibitors of Fasciola gigantica thioredoxin glutathione reductase

Fascioliasis is caused by the helminth parasites of genus Fasciola. Thioredoxin glutathione reductase (TGR) is an important enzyme in parasitic helminths and plays an indispensable role in its redox biology. In the present study, we conducted a structure‐based virtual screening of natural compounds against the Fasciola gigantica TGR (FgTGR). The compounds were docked against FgTGR in four sequential docking modes. The screened ligands were further assessed for Lipinski and ADMET prediction so as to evaluate drug proficiency and likeness property. After refinement, three potential inhibitors were identified that were subjected to 50 ns molecular dynamics simulation and free energy binding analyses to evaluate the dynamics of protein‐ligand interaction and the stability of the complexes. Key residues involved in the interaction of the selected ligands were also determined. The results suggested that three top hits had a negative binding energy greater than GSSG (−91.479 KJ · mol−1), having −152.657, −141.219, and −92.931 kJ · mol−1 for compounds with IDs ZINC85878789, ZINC85879991, and ZINC36369921, respectively. Further analysis showed that the compound ZINC85878789 and ZINC85879991 displayed substantial pharmacological and structural properties to be a drug candidate. Thus, the present study might prove useful for the future design of new derivatives with higher potency and specificity.

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis.

Comprehensive analysis of the catalytic and structural properties of a mu-class glutathione s-transferase from Fasciola gigantica

Glutathione S‒transferases (GSTs) play an important role in the detoxification of xenobiotics. They catalyze the nucleophilic addition of glutathione (GSH) to nonpolar compounds, rendering the products water-soluble. In the present study, we investigated the catalytic and structural properties of a mu-class GST from Fasciola gigantica (FgGST1). The purified recombinant FgGST1 formed a homodimer composed of 25 kDa subunit. Kinetic analysis revealed that FgGST1 displays broad substrate specificity and shows high GSH conjugation activity toward 1-chloro-2,4-dinitrobenzene, 4-nitroquinoline-1-oxide, and trans-4-phenyl-3-butene-2-one and peroxidase activity towards trans-2-nonenal and hexa-2,4-dienal. The FgGST1 was highly sensitive to inhibition by cibacron blue. The cofactor (GSH) and inhibitor (cibacron blue) were docked, and binding sites were identified. The molecular dynamics studies and principal component analysis indicated the stability of the systems and the collective motions, respectively. Unfolding studies suggest that FgGST1 is a highly cooperative molecule because, during GdnHCl-induced denaturation, a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed. The protein is stabilized with a conformational free energy of about 10 ± 0.3 kcal mol−1. Additionally, the presence of conserved Pro-53 and structural motifs such as N-capping box and hydrophobic staple, further aided in the stability and proper folding of FgGST1.

Salt-regulated reversible fibrillation of Mycobacterium tuberculosis isocitrate lyase: Concurrent restoration of structure and activity

Protein fibrillation is associated with a number of neurodegenerative diseases. Nevertheless, several proteins not related to disease can also form fibrils in vitro under specific conditions. In the present study, we demonstrate the reversible fibrillation of a globular protein that is modulated by salt under physiological pH. Mycobacterium tuberculosis Isocitrate lyase (MtbICL) is a crucial enzyme involved in the glyoxylate shunt and a potential drug target against M. tuberculosis infection. Under physiological pH, the enzyme self-assembles into a fibrillar structure in the absence of salt in vitro. The mature fibrillar structure of MtbICL is dynamic and restores its tetrameric structure as well as activity with the addition of salt. The kinetics of fibril formation was investigated spectroscopically using 8-Anilinonaphthalene-1-sulfonic acid (ANS). Further, Transmission electron microscopy (TEM) and Atomic force microscopy (AFM) imaging also confirmed the formation of elongated fibrils in the absence of salt. The results indicate the balance between stabilizing forces and the localized electrostatic repulsions destabilizing the tetrameric MtbICL is adjusted via ion shielding. Our result is in congruence of the hypothesis that amyloid formation is an intrinsic property of most, if not all natural proteins under an appropriate set of conditions.