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...
