243 - Tsa1 and Tsa2 Are Two Highly Similar 2-Cys Prx from Yeast That Display Remarkable Differences in Their Structural Switches

Abstract

Typical 2-Cys Peroxiredoxins (2-Cys Prxs; AhpC-Prx1) are enzymes that reduce hydroperoxides with extraordinary rates and are involved in diseases such as cancer, neurodegeneration and Alzheimer. The active site is composed of a catalytic triad, containing the peroxidatic cysteine (C P ), an Arg, and a Thr (or Ser). After oxidation of C P , an intermolecular disulfide is formed between C P and a second cysteine residue (resolving cysteine – C R ). During catalysis, 2-Cys Prxs switch between dimers and decamers (pentamer of dimers) or even to higher molecular weight species, depending on redox state among other factors. Additionally, a conformational change in the active site loop (partial unfolding of the α helix C P ) is required for the generation of an intermolecular disulfide. Therefore, during catalytic cycle, 2-Cys Prx alternate between fully folded (FF - reduced enzyme), and locally unfolded (LU) states, when disulfide bond can be formed. The analysis of all available 2-Cys Prx structures in FF revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic Thr of a dimer and an aromatic residue of adjacent dimer. However, none of the structures with the natural substitution of Thr by Ser display this feature. Structural analysis of 2-Cys Prx containing Thr (but not Ser) in the active site indicated that this residue destabilizes the decamer in the oxidized state possibly due to steric hindrance. This hypothesis was experimentally confirmed using two homologs yeast 2-Cys Prx (Tsa1 and Tsa2). In spite of possessing a high degree of similarity (~86%), Tsa1 presents a Thr in the active site, while Tsa2 presents a Ser. Size exclusion chromatography demonstrated that Tsa1 decamer (but not Tsa2) was destabilized by the oxidation. Additionally, mutants of Tsa1 which were observed mainly as dimers also displayed a decreased catalytic activity. Taken all the data together, we believe that oligomeric state of these enzymes may be related to regulatory pathways.