Structural Insights Into The Catalytic Mechanism Of Yeast Glutathione S-transferase Gtt2 And Methionine-S-sulfoxide Reductase Mxr1

(Ⅰ) Many xenobiotics have cytotoxic and/or genotoxic properties, and can damage cells in various ways. To eliminate toxic xenobiotics, living organisms have evolved a series of sophisticated detoxification strategies. In general, the detoxification process can be described as a three-phase reaction. In phase I, xenobiotics are activated by introduction of reactive functional groups. In phase II, they are neutralized by conjugation to chemical constituents through the reactive groups. In phase III, the conjugated xenobiotics are pumped out from cells after being metabolized via downstream pathways and eventually eliminated.The glutathione S-transferases (GSTs) (EC 2.5.1.18) play a key role in phase II of enzymatic detoxification. These enzymes catalyze the reaction of xenobiotics with the thiolate group of glutathione (GSH), thereby neutralizing their electrophilic sites and raising the water-solubility of the products. Eukaryotes usually contain multiple GST paralogs with different catalytic activities, and a wide range of cellular functions. In mammals, cytosolic GSTs are dimeric proteins that are grouped into eight main-strain classes, primarily by sequence alignment: Alpha, Kappa, Pi, Mu, Theta, Zeta, Sigma and Omega. In addition, studies in non-mammalian species have revealed several new classes such as theΒeta class of bacteria, the Phi and Tau classes in plants, and the Delta class in insects. Fungi also possess several GSTs, but most cannot be grouped into the pre-existing classes. In addition to the sequence-based classification, the nomenclature of cytosolic GSTs can refer to the essential catalytic residue. All cytosolic GSTs of known structure are classified into one of three catalytic types, either Tyr-, Ser- or Cys-type, depending on the residue that drives the conjugation of GSH to a xenobiotic through lowering the pKa of GSH, and stabilizing the thiolate anion via a hydrogen bond. Mutations of the essential residue usually results in a substantial, if not complete, inactivation of the GST.In the yeast Saccharomyces cerevisiae, seven proteins possess GST activity (Gtt1 and 2, Gto1, 2 and 3 and Grx1, 2). However, the structures of Grx1 and Grx2 reveal that they are not bona fide GSTs, but only correspond to the GST N-terminal domain. Of the five other homologous proteins, Gto1, 2 and 3 are classified into the Omega GST class, while Gtt1 and 2 are not categorized into any existing classes. Although the physiological and biochemical functions of these five yeast GSTs have been extensively studied, no three-dimensional (3-D) structures have been reported. We began a systematic characterization of the yeast GSTs by determining the crystal structures of Gtt2 in apo form at 2.23 (A|°), and in two ligand-bound forms at 2.20 and 2.10 (A|°). Gtt2 is distinct from the three classic catalytic types, because a water molecule fixed by two residues in the C-terminal domain is responsible for the activity. Moreover, only glycine and alanine are favored at the N-terminus of helixα1 because of the stereo-hindrance. These results enabled us to define a novel catalytic cytosolic GST type, which is called the atypical-type. (Ⅱ) Thioredoxins (Trxs) are ubiquitous small thiol-disulfide exchange proteins which are involved in many important cellular processes such as reduction of methionine sulfoxide, ribonucleotide, and peroxide. These proteins have a highly conserved active site of CXXC motif. During reaction, the first Cys attacks the intramolecular disulfide bond in the substrate protein, accompanying with the formation of an intermolecular disulfide intermediate. This mixed disulfide is subsequently attacked by the second Cys, resulting in the release of reduced substrate protein and the oxidized Trx.Methionine is one of the most sensitive amino acid residues subject to oxidation. It can be readily oxidized to methionine sulfoxide (Met-SO) as a mixture of two enantiomers at the sulfoxide moiety (Met-S-SO and Met-R-SO), by various reactive oxygen or nitrogen species. Oxidation of the methionines on protein surfaces would cause some lethal effects to the cells and accelerate the aging process. However, both in vitro and in vivo, a group of enzymes called methionine sulfoxide reductases (Msrs, EC 1.8.4.6) can use thioredoxin (Trx) as the electron donor to regenerate the oxidized proteins, by reducing Met-SO to methionine. Recently, the glutathione/glutaredoxin system has been discovered that it also could act as the electron donor for Met-SO reduction. This protective mechanism has been shown to play a significant role in elongating the lifespan of yeast, insects and mammals.To date, three Msr families have been reported. MsrA and MsrB are classic Msrs that regenerate the proteinous Met-S-SO and Met-R-SO, respectively. They also could regenerate the corresponding free Met-SO. A series of human disease-related proteins have been identified as substrates of MsrA and MsrB, such as calmodulin, HIV-2 protease and alpha-1-proteinase inhibitor. However, fRMsr is a recently discovered Msr which is exclusively responsible for the reduction of free Met-R-SO, but not the proteinous ones. Despite that the three families have distinct differences in origin, structure, substrate specificity and species distribution, they basically share a similar catalytic mechanism. The mechanism involves the oxidation of the catalytic cysteine to a sulfenic acid intermediate, followed by the formation of an intramolecular disulfide bond, and the final regeneration process driven by Trx or other reductants.Structures of MsrA from seven different species are currently reported. The core structures of these MsrA are very similar and could be well superimposed. This is regardless of the method of structure determination, either crystallography or NMR spectroscopy. The major differences are involved in the loops around the active site, especially the C-terminal loop, which is supported by the electrostatic analysis and distance measurements between the two catalytic cysteines. It is also suggested that these conformational changes would facilitate the formation of an intramolecular disulfide bond and the exposure of a hydrophobic patch for Trx interaction.The yeast Saccharomyces cerevisiae encodes three Msrs (Mxr1/MsrA, Mxr2/MsrB and Ykg9/fRMsr). Although the mechanism of the Msrs has been extensively studied, their interactions with diverse substrate proteins and a universal Trx remain unknown. Therefore, we have systematically characterized the yeast Msr-Trx complexes by determining the crystal structures of Mxr1 in its reduced form at 2.04 (A|°), in a dimeric oxidized form at 1.90 (A|°), and in a Trx2-complexed form at 2.70 (A|°). Superposition of these structures have revealed three highly flexible loops. These loops undergo drastic conformational changes and may be responsible for the enzyme's substrate diversity. Additionally, interface analysis of Mxr1-Trx2 along with data from previously reported Trx-protein complexes, we have found a new mode for Trx-involved complexes which undergo drastic conformational changes. These findings would be helpful for the prediction of potential interfaces on the Trx substrate proteins.