Approved Abstracts


Author(s): Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC; Universidade Federal de Santa Catarina, UFSC;
Presenter: Luiz Otávio de Barros Vilas Bôas

Specific tools for environmental biomonitoring, such as biomarkers, are necessary for the identification, assessment, and remediation of the anthropic impact on aquatic environments. Enzymes from phase II biotransformation systems, such as Glutathione S-transferases (GST), are among the classic biomarkers. The present study aimed to structurally and functionally characterize GST Omega (GSTO) from the Pacific oyster Crassostrea gigas. The GSTO is known to play a role in the process of antioxidant defenses and biotransformation of xenobiotics of the most varied types in many species. C. gigas is the most cultivated oyster in the world. Due to their filtering and sessile characteristics, and wide geographic distribution, it has been widely used in biomarker studies. In oysters, significant changes were observed in GSTO gene transcription and enzymatic activity when comparing aquaculture and natural sites; different salinities; pH, and after exposure to hydrocarbons. Despite these studies, little is known about the GST family in C. gigas, regarding functional mechanisms of different classes and isoforms. CgGSTO2.1 sequence was obtained from the NCBI database (accession number XP_011429380.2). A tridimensional model was constructed using the I-TASSER and HADDOCK platforms. The predicted structure was used to estimate the conditions for further in vitro and in silico experiments. To evaluate binding ligand properties, molecular docking assay was performed by using SwissDock platform. The Glutathione (GSH), Glutathione S-(2,4 dinitrobenzene) (GDN), and Dehydroascorbate (DHA), were tested as substrates catalyzed by Omega isoform. The obtained positions were evaluated by the binding affinity (ΔG), ligand position compared to a control crystal structure and weak interactions between ligand and residues. Furthermore, the recombinant CgGSTO2.1 isoform gene was constructed in the pET-19b vector and expressed in BL21 (DE3) pLys S Escherichia coli strain. The recombinant protein was purified by nickel affinity chromatography. Regarding in silico analysis, the CgGSTO2.1 3D structure presented a highly conserved G-site compared to other species. Particularly, a non-conserved Cys residue was identified in C.gigas omega isoform. Molecular docking showed that GSH binds the G-site and interacts with the expected catalytic residues (K55, Y79, E80) with ΔG -9,0 kcal/mol, which is similar to the values from redocking of human GST Omega (ΔG -11.6 kcal/mol), available on Protein data bank (PDB-1EEM.1). When GSH-dinitrobenzene (GDN) was tested, the free energy estimation reached similar values (ΔG - 6.8 kcal/mol). These data are close to those obtained with the redocking of the crystallographic structure from Trametes versicolor Omega isoform (PDB-6F4F) used as a positive control (ΔG -12.8 kcal/mol). In addition, our data suggest that C.gigas GST Omega isoform may interact with DHA via Tyr 79, besides lower affinity (ΔG -5.4 kcal/mol). A conserved Tyr residue involved in DHA binding was also observed in the human crystal structure used as control (PDB-3VLN), with control redocking value of ΔG -6.16 kcal/mol). Recombinant CgGSTO2.1 protein was expressed and purified, and in vitro analysis with DHA, 2,4-dinitrobenzene (CDNB), 4-hydroxynonenal (4-HNE), and ethacrynic acid (EA) are being carried out to estimate the catalytic activity (Km, kcat and Vmax). These results will be compared to in silico analysis that suggest the tested ligands are potential substrates for the CgGSTO2.1.

Keywords: Biomarker; Molecular docking; Bivalve mollusks




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