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

Jupitara Kalita,Rajanish Giri,Rohit Shukla,Harish Shukla,Kundlik Gadhave,Timir Tripathi

doi:10.1038/s41598-017-17678-3

Jupitara Kalita, Rajanish Giri + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-17678-3

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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.

Highlights

Fascioliasis, a neglected tropical disease, is caused by the food-borne trematodes Fasciola hepatica and Fasciola gigantica
Helminth Glutathione S‒transferases (GSTs) are an important target for chemotherapeutic and vaccine development
The phylogenetic tree showed that FgGST1 shared the evolutionary clade with FhGST1-mu and is very distinct from the human GSTs (Supplementary Figure S1)

Summary

Multiple sequence alignment showed that

FgGST1 has the highest sequence identity with FhGST (96.36%). FgGST1 showed 76.39%, 75%, 72.69%, 68.98%, 64.78%, 60.19%, 55.09%, 48.61%, 46.79%, 44.19%, 44.50%, 42.86%, 42.40%, and 46.3% identity with FhGST47, FhGST-mu, FhGST7, P. westermani, C. sinensis, S. mansoni, P. westermani, D. japonica, S. solidus, E. multilocularis, E. multilocularis-mu[1], T. solium, E. granulosus, and H. sapiens GST, respectively (Fig. 1). The predicted FgGST1 model was superimposed with FhGST structure that showed an excellent RMSD value of 0.152 Å for 216 atom pairs (Fig. 6A). The FgGST1-CB complex was stabilized by three hydrogen bonds with Tyr[7], Leu[13] and eight hydrophobic interactions with Trp[8], Leu[10], Asn[54], Pro[56], Gly[205], Trp[206], and His[207] from the chain-A residues (Fig. 7B). FgGST1 and FgGST1-CB complexes showed an average RMSD value of 0.34 and 0.50 nm, respectively (Fig. 8A) These values suggest that the modeled FgGST1 structure was more stable as compared to the FgGST1-CB complex. From the 2D projection plot (Fig. 8F), it was observed that FgGST1-CB complex is more stable as it showed some stable cluster and occupied less phase space as compared to the modeled FgGST1 structure. In the presence of GdnHCl, the nucleophilicity of the GS– anion is reduced and the activity of the enzyme is decreased

Polar salvation energy

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