Structural And Enzymatic Insights Into Saccharomyces Cerevisiae Alkyl Hydroperoxide Reductase Ahp1

Reactive oxygen species (ROS) are highly reactive metabolites of oxygen that contain hydrogen peroxide (H2O2), alkyl peroxides (ROOH), hydroxyl radical (HO·), and the superoxide anion (O2). The accumulation of ROS had been linked to several diseases, such as Alzheimer’s and Parkinson’s diseases, cancer, and aging.Peroxiredoxin (Prx, EC1.11.1.15) is a family of peroxidases that protect cells against ROS, such as hydrogen peroxide and alkyl peroxides. Prx reduces peroxides with redox-active cysteines. Two cysteines called peroxidatic cysteine (CP) and resolving cysteine (CR) are involved in the catalytic cycle of2-Cys Prx. CP is the conserved residue that attacks substrates peroxides, and is usually located at the N-terminal region of Prx. CR is the residue that resolves the oxidized CP and results in the formation of a CP-CR disulfide bond. However, The CR residue is omitted for1-Cys Prx. Although the catalytic mechanisms of Prxs have been extensively studied, insights into their turnover by thioredoxins remain unclear.The alkyl hydroperoxide reductase Ahpl is one of five Prxs (Tsal, Tsa2, Prx1, Dot5and Ahpl) in the yeast Saccharomyces cerevisiae. The AHP1null mutant yeast strain is sensitive to tert-butyl hydroperoxide (t-BOOH). Sequence comparison suggests that Ahpl is a member of the Prx5subfamily. A previous report showed that Ahpl was a typical2-Cys Prx with an intermolecular disulfide bond involving the Cp (Cys62) and CR (Cys120). However, our enzymatic assays and bioinformatics indicated that the Cp and CR were Cys62and Cys31, respectively. Furthermore, the structure of oxidized Ahpl at2.40A resolution showed an intermolecular disulfide bond between Cys62and Cys31. Thus, Cys31, but not the previously reported Cys120, should be the CR. Comprehensive analyses demonstrated that Ahp1represents a novel group of typical2-Cys Prxs of four unique features. First, the CR is located at the N-terminal region and preceding the CP in the primary sequence. Second, the intermolecular CP-CR disulfide bond crossing the A-type dimer interface. Third, despite it is structurally similar to members of atypical2-Cys Prxs and1-Cys Prxs, it is mimicking members of typical2-Cys Prxs from the mechanistic point of view. Four, the members of this group are distributed in the Prx5subfamily and are found in some fungi and bacteria. Reaction kinetics assays demonstrated the positive cooperativity of the substrate t-BOOH binding to Ahp1homodimer at a Hill coefficient of approximately2, which enabled Ahpl to eliminate hydroperoxide at much higher efficiency. This provided a new evidence for the evolutionary advantage of Ahp1dimerization. We determined the crystal structure of Ahpl in reduced form at2.91A resolution. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing Cp and Cr. The two turns of the helix a2(Ser59-His66) of the reduced Ahpl will be unraveled, upon oxidation, into loop conformation, and shift towards the CR, whilst the segment juxtaposed to CR (Gln23-Lys32) moves inwards, resulting in the approach of CP and CR for the formation of the intermolecular disulfide bonds. This oxidized form is inactivated, and subject to the attack of Trx2. To understand the process of regeneration, by using cross-linking mediated by5,5’-dithiobis-(2-nitrobenzoic acid)(DTNB), we identified that Cys31of Ahp1was attacked by Cys31of Trx2to form an intermediate complex of Ahpl-Trx2via an intermolecular disulfide. We purified this complex and determined the structure at2.10A resolution. It showed that the disulfide bonds of Ahpl homodimer are recognized and attacked by Trx2from both sides, resulting in the formation of intermediate disulfide bonds between Cys31of Ahpl and Cys31of Trx2. The loop Asp29-Met33of Ahpl is recognized by Trx2via four conserved main-chain hydrogen bonds and one side-chain hydrogen bond. Ahp1-Trx2complex is further stabilized via hydrophobic interactions mainly contributed by solvent-accessible hydrophobic patches of Trx2(Trp30-Pro33around the redox center and residue Met73) and two residues of Ahpl (Met33and Pro60). Based on this complex structure, we deduced that, after the attack by Cys34of Trx2on the mixed disulfide bond between Cys31of Ahpl and Cys31of Trx2, a regenerated Ahp1will depart from an oxidized Trx2(Cys31-Cys34) for another catalytic cycle. To date, this is the first Trx-complexed structure of a peroxidase or a Trx-like protein. The complex structure shows the electron transfer from the electron donor Trx to the receptor Prx.Recent studies indicated that residue Lys32of Ahpl can be covalently conjugated to ubiquitin-related modifier, Urm1, in S. cerevisiae. However, the function of urmylated Ahpl remains unknown. Our results demonstrated that residue Cys31plays a crucial role in the peroxidase activity of Ahp1. Furthermore, residue Lys32is involved in the interface between Ahp1and Trx2. Thus, will urmylation of Lys32affect the catalysis and the regeneration process of Ahp1? We found that the regeneration efficiency of Ahpl driven by Trx2was significantly decreased upon the mutation of Lys32to Ala or Glu, and that the regeneration efficiency of K32E mutant was about1/3of wild-type. On the other hand, covalent conjugation of Unnl to the Lys32of Ahpl might introduce steric hindrance. Therefore, we suggest that urmylation of Lys32would decrease or abolish the peroxidase activity of Ahpl, and make the oxidized Ahpl a molecule to pass the signal of oxidation.