Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders. P rotein-bound methionine residues are among the most susceptible to oxidation by reactive oxygen species (ROS), resulting in formation of methionine sulfoxide [Met(O)] residues. However, this modification can be repaired by methionine sulfoxide reductase (MsrA), which catalyzes the thioredoxindependent reduction of free and protein-bound Met(O) to methionine, both in vitro (1) and in vivo (2). Bacteria and yeast cells lacking the msrA gene show increased sensitivity to oxidative stress and lower survival rates (3, 4), with yeast showing accumulation of high levels of both free and protein-bound Met(O) (2, 4). In addition, overexpression of the MsrA enzyme in human T cells prolongs their life under conditions of oxidative stress (4). Because methionine residues are particularly susceptible to oxidation by ROS, MsrA could have at least three important functions in cellular metabolism: (i) as an antioxidant enzyme that scavenges ROS by facilitating the cyclic interconversion of methionine͞protein-methionine residues between oxidized and reduced forms (2); (ii) as a repair enzyme by keeping critical methionine residues in their reduced form; and (iii) as a regulator of critical enzyme activity through cyclic interconversion of specific methionine residues between oxidized and reduced forms (5, 6). Escherichia coli and Saccharomyces cerevisiae both contain at least two Msrs. One (MsrA) is able to reduce both free and protein-bound Met(O), and the other can reduce only free Met(O). The MsrA protein is highly expressed in liver, kidney, pigment epithelial cells of the retina, macrophages, cerebellum, and brain neurons (7). These tissues͞ cells are sensitive to oxidative stress damages. Therefore, abolishing the MsrA enzyme could lead to loss of antioxidant defense, resulting in enhanced oxidative damage and a decreased lifespan. To investigate the possible role of MsrA as an antioxidant in mammals and its possible influence on lifespan, we created a strain of mouse lacking the MsrA protein. Materials and ...

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The Escherichia coli peptide methionine sulfoxide reductase gene (msrA) encodes a single-subunit polypeptide of 212 amino acid residues (M. A. Rahman, H. Nelson, H. Weissbach, and N. Brot, J. Biol. Chem. 267:15549-15551, 1992). RNA blot analysis showed that the gene is transcribed into an mRNA of about 850 nucleotides. The promoter region was characterized, and the transcription initiation site was identified by primer extension. The synthesis of the MsrA protein increased about threefold in a growth-phase-dependent fashion. In an attempt to define the in vivo role of msrA, a chromosomal disruption was constructed. This mutant was more sensitive to oxidative stress, suggesting that oxidation of methionine in proteins plays an important role in oxidative damage.The enzyme peptide methionine sulfoxide reductase (MsrA) catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine (1,5,12). The presence of Met(O) residues in proteins may arise during aerobic metabolism as a consequence of the oxidation of methionine by such reagents as hydrogen peroxide, hydroxyl radicals, and hypochlorite and superoxide ions (reviewed in reference 4). It has also been reported that oxidized linolenic acid is capable of oxidizing methionine residues in proteins in vitro (20). In many cases, the oxidation of a specific methionine residue leads to the loss of biological activity (4), which can be restored upon incubation of the oxidized protein with MsrA (1,5,12,17,27).The gene for the enzyme from Escherichia coli has recently been cloned and sequenced (24). The coding region of the gene is 636 nucleotides long and encodes a single-subunit protein with a calculated molecular weight of 23,316. In addition, the reductase has been overexpressed in E. coli and purified to homogeneity (23). In this report, we examine the regulation of expression of this gene and show that an msrA mutant is more sensitive to oxidative damage than wild-type cells. MATERIALS AND METHODS Materials.Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, Mass.). Avian myeloblastosis virus reverse transcriptase was from Promega (Madison, Wis.). Radioisotopes were from either Amersham (Arlington Heights, Ill.) or Dupont/NEN (Boston, Mass.). Immobilon N transfer membranes were from Millipore (Bedford, Mass.). All chemicals were from Sigma (St. Louis, Mo.) or Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Purified MsrA and antiserum against the protein were prepared as previously described (23).Northern (RNA) analysis. Total RNA was isolated as described previously (3) from 100-ml cultures (A 600 of 0.6) of either E. coli XL1-Blue or E. coli XL1-Blue/pAR100, which contains a plasmid bearing a genomic copy of the msrA gene (24). The coding region of the gene was amplified by PCR using two synthetic primers. The DNA was purified by agarose gel electrophoresis and the GeneClean (Bio 101, La Jolla, Calif.) procedure. Five hundred nanograms of the DNA was used for random prim...