Characterization of the mechanism of thioredoxin-dependent activation of γ-glutamylcyclotransferase, RipAY, from Ralstonia solanacearum.

ABSTRACT

A class II ChaC protein, RipAY, from phytopathogenic bacterium, Ralstonia solanacearum exhibits γ-glutamylcyclotransferase (GGCT) activity to degrade intracellular glutathione in host cells upon its interaction with host thioredoxins (Trxs). To understand the Trx-dependent activation of RipAY, we constructed various deletion mutants of RipAY and found the determinant region for GGCT activation in the N- and C-terminal sequences of RipAY by analyzing their yeast growth inhibition activity and the interaction with Trxs. Mutational analysis of the active site cysteine residues of Arabidopsis thaliana Trx-h5 (AtTrx-h5), one of the most efficiently stimulating Trxs, revealed that each active site cysteine residue of AtTrx-h5 contributes to efficient RipAY-binding and -activation activity. We also estimated that RipAY and AtTrx-h5 form a complex at a 12 M ratio. Furthermore, we found that the constitutive GGCT activity of Gcg1, a yeast class I ChaC protein, is also stimulated by yeast Trx1. These results indicate that class I ChaC proteins can sense the intracellular redox state and interact with Trxs to promote more efficient degradation of glutathione and regulate intracellular redox homeostasis. We hypothesize that RipAY acquired a more efficient and specific Trx-dependent activation mechanism to activate its GGCT activity only in the host eukaryotic cells during the evolution.