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

Abstract

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.

Monomeric Camelus dromedarius GSTM1 at low pH is structurally more thermostable than its native dimeric form

“…GST class μ (GSTM; 51.5 kDa) is a homodimeric protein [19,20]. Each subunit comprises two structural domains: the N-terminal domain (NTD) and the C-terminal domain (CTD).…”

The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica

“…Thus, to determine if rFhGST-Mu5 is an isoform with 'low affinity' for GSH, the specific activity was determined with the model substrate CDNB (Habig et al 1974). The specific activity of rFhGST-Mu5 was significantly lower than that recorded for the previously known 4 Mu class GSTs from F. hepatica (Kalita et al 2017;Salvatore et al 1995). This lower affinity may be correlated with the lower sequence homology and the more distant grouping of FhGST-Mu5 in phylogenetic modelling aligning closer to schistosome Mu class GSTs rather than the previous four F. hepatica Mu class.…”

Targeting Nucleotide Binding Domain of Multidrug Resistance-associated Protein-1 (MRP1) for the Reversal of Multi Drug Resistance in Cancer

“…MD simulations were performed in an in-house supercomputer using GROMACS 4.6.5 47 , 48 with GROMOS 9653a6 force field and SPC216 water model as previously mentioned 49 – 52 . A total of three systems (one for apo-protein and two for protein-ligand complex) were created for 100 ns MDS.…”

“…To understand the conformational changes, root mean square deviation (RMSD) and radius of gyration (Rg) of all backbone Cα atoms were computed with respect to the native structure at 300 K. The structural stability of the proteins was defined in terms of root mean square fluctuations (RMSF) and H-bond interaction to provide direct evidence of protein ligand stability. The RMSD and RMSF were calculated using g_rms and g_rmsf tools as mentioned earlier 52 while the H-bonds were and principal component analysis (PCA) were carried out by using g_hbond, g_covar and g_anaeig tool of GROMACS package. The trajectories were analyzed using Chimera 1.10.2, and the graphs were plotted by Origin 6.0 software.…”