Catalytic Thr or Ser Residue Modulates Structural Switches in 2-Cys Peroxiredoxin by Distinct Mechanisms

Abstract

Typical 2-Cys Peroxiredoxins (2-Cys Prxs) reduce hydroperoxides with extraordinary rates due to an active site composed of a catalytic triad, containing a peroxidatic cysteine (CP), an Arg, and a Thr (or Ser). 2-Cys Prx are involved in processes such as cancer; neurodegeneration and host-pathogen interactions. During catalysis, 2-Cys Prxs switch between decamers and dimers. Analysis of 2-Cys Prx structures in the fully folded (but not locally unfolded) form revealed a highly conserved, non-conventional hydrogen bond (CH-π) between the catalytic triad Thr of a dimer with an aromatic residue of an adjacent dimer. In contrast, structures of 2-Cys Prxs with a Ser in place of the Thr do not display this CH-π bond. Chromatographic and structural data indicate that the Thr (but not Ser) destabilizes the decamer structure in the oxidized state probably through steric hindrance. As a general trend, mutations in a yeast 2-Cys Prx (Tsa1) favoring the dimeric state also displayed a decreased catalytic activity. Remarkably, yeast naturally contains Thr-Ser variants (Tsa1 and Tsa2, respectively) with distinct oligomeric stabilities in their disulfide states.

Saccharomyces cerevisiae Peroxiredoxins in Biological Processes: Antioxidant Defense, Signal Transduction, Circadian Rhythm, and More

“…In fact, the analysis of the structure revealed an interaction of the Thr from the active site motif, at the dimer-dimer interface of the decamer. Recently, using different methodological approaches as site-directed mutagenesis, biochemical approaches, size exclusion chromatography, and structural analysis, we have demonstrated that a slight difference in the PXXXT(S)XXC P is involved in decamers to dimers transitions [10]. While Tsa1 possess a Thr residue embedded in the conserved motif, in Tsa2, the Thr is naturally substituted by a Ser (Figure 4).…”

“…Despite that the basic functional unit of the typical 2-Cys Prx is represented by a α(2) homodimer, studies using the Tsa1 and Tsa2 isoforms from S. cerevisiae revealed that this oligomeric state presents low peroxidase activity, and the highest reactivity of the typical 2-Cys Prx is reached when these proteins are found in a ring-shaped α2(5) decamers (association of five homodimers; Figure 3A). It is believed that the alternation between these two quaternary structures is responsible for the modulation of their peroxidase activity and may be involved in signal transduction ( Figure 3B) [10]. Additionally, the typical 2-Cys Prx enzymes may also present other oligomeric states that will be discussed posteriorly.…”

“…The latter ones have been subject of intense studies since works involving kinetic approaches indicate that the Prxs decompose more than 90% of cellular hydroperoxides [8,9]. Additionally, to exert their biological functions, several Prxs are able to perform amazing structural switches, revealing an intricate puzzle among protein structure and function [10][11][12].…”

“…The Prx enzymes are able to decompose hydroperoxides without any prosthetic group, but using only a highly reactive cysteine residue named peroxidatic cysteine (C P ) [5,22]. All the Prxs described to date present a conserved motif containing the C P (PXXXT/SXXC P ), which is oxidized to cysteine sulfenic acid (C P -SOH) after hydroperoxide reduction [10]. This enzyme family is very heterogeneous, and different classifications have been proposed; the most currently used one subdivides these proteins in three large subclasses,1-Cys Prx, typical 2-Cys Prx, and atypical 2-Cys Prx, based in the number of cysteines involved in catalytic cycle and structural aspects (Figure 1).…”

“…The C P thiolate is stabilized by polar interactions with a threonine (or a serine, in some cases) and an arginine residue ( Figure 3C). These three residues (Thr, C P , and Arg) are named catalytic triad and are widely conserved among all Prxs described to date [10]. During catalysis, a guanidine group of the Arg residue is able to perform a hydrogen bond with the proximal oxygen (O) of the hydroperoxide, allowing the nucleophilic attack of the C P over the hydroperoxide [24].…”