Thiol–disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase

Abstract

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thioldisulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol-disulfide exchange reactions via Cys-227, DTT, or thioredoxin.oxidation ͉ cysteine ͉ thioredoxin ͉ Alzheimer's disease ͉ aging

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson's disease-related insults

“…Two active-site cysteine residues play key roles in MsrA activity. Cysteine 72 carries out a nucleophilic attack at the sulfur atom of the methionine sulfoxide substrate, leading to the formation of a covalent intermediate, whereas cysteine 218 attacks cysteine 72 to trigger breakdown of the covalent complex [24].…”

“…Recombinant MsrA was prepared using a method adapted from a previous study [24]. Cells of the BL21(DE3) strain of Escherichia coli were transformed with the pET28b-MsrA construct by electroporation.…”

“…Primary cultures were untransduced or transduced with lentivirus or adenovirus encoding wild-type MsrA or two catalytically inactive mutants, C72S or C218S [24], and subsequently treated with rotenone. The relative number of TH + neurons was greater in cultures transduced with wild-type MsrA virus compared to untransduced cells or cells infected with virus encoding C72S or C218S ( Figure 1C,D).…”

“…Two active-site cysteine residues play key roles in MsrA activity. Cysteine 72 carries out a nucleophilic attack at the sulfur atom of the methionine sulfoxide substrate, leading to the formation of a covalent intermediate, whereas cysteine 218 attacks cysteine 72 to trigger breakdown of the covalent complex [24].The results of studies in MsrA-knockout mice [25] and in flies over-expressing MsrA in neural tissues [26] suggest that down-or up-regulation of MsrA is associated with decreased or increased longevity, respectively. MsrA activity decreases with age in rats [27], and this decrease coupled with the resulting age-related increase in oxidative stress may explain in part why the incidence of PD increases with age [1,2].…”

“…A lentiviral construct encoding A53T was generated as described previously [29][30][31]. The construct pET28b-MsrA, which contains the gene encoding bovine MsrA including the N-terminal mitochondrial targeting sequence [24,32], was provided by Dr. Todd Lowther (Wake Forest University). The MsrA gene was amplified from this construct by PCR and subcloned as a BamH I-Xho I fragment into pENTR1A to generate the construct, pENTRMsrA.…”