Structural and functional analysis of the bifunctional thioredoxin glutathione reductase -TGR- from the white shrimp Penaeus vannamei, and redox responses under hypoxia.

Glutathione (GSH) plays a key role in cellular homeostasis. There are two major enzymatic pathways in invertebrates to reduce oxidized glutathione (GSSG): 1) glutathione reductase (GR) and 2) thioredoxin glutathione reductase (TGR). In some insects, thioredoxin reductase (TR), reduces GSSG non-enzymatically via thioredoxin. The search for these enzymes in crustaceans has recently become a focal point to better understand redox homeostasis under stress conditions. This study aimed to characterize TGR in the shrimp Penaeus vannamei and to evaluate gene expression, GR activity, GSH and GSSG content, and thiobarbituric acid reactive substances (TBARS) in the gills following short-term hypoxia and reoxygenation. The TGR coding sequence (CDS) is 1989 bp and encodes 662 amino acids; it is a selenoprotein with conserved motifs for selenocysteine insertion, GSH, thioredoxin, and FAD/NADPH binding pockets, classifying this enzyme as a TGR. The deduced amino acid sequence contains a 66-residue N-terminal mitochondrial targeting sequence. Phylogenetic analysis showed that PvTGR is close to other invertebrates and shares a common ancestry with the TGR from Platyhelminthes. Expression of the TGR gene was induced during hypoxic stress and decreased upon reoxygenation, demonstrating its involvement in the stress response. The GSH/GSSG ratio decreased in a time-dependent manner, and TBARS returned to initial normoxia levels during reoxygenation after 6 h hypoxia. The identification and characterization of TGR are key to future studies on the effects of stress on the responses of GSH-metabolism-involved enzymes in Crustacea.