Purification and Characterization of Methionine Sulfoxide Reductases from Mouse and Staphylococcus aureus and Their Substrate Stereospecificity Jackob Moskovitz,* ,1 Vineet K. Singh,† Jesus Requena,* Brian J. Wilkinson,† Radheshyam K. Jayaswal,† and Earl R. Stadtman* *Laboratory of Biochemistry, National Institute of Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and †Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois 61790 Received November 1, 2001 Many organisms have been shown to possess a me- thionine sulfoxide reductase (MsrA), exhibiting high specificity for reduction the S form of free and protein- bound methionine sulfoxide to methionine. Recently, a different form of the reductase (referred to as MsrB) has been detected in several organisms. We show here that MsrB is a selenoprotein that exhibits high speci- ficity for reduction of the R forms of free and protein- bound methionine sulfoxide. The enzyme was par- tially purified from mouse liver and a derivative of the mouse MsrB gene, in which the codon specifying sel- enocystein incorporation was replaced by the cystein codon, was prepared, cloned, and overexpressed in Escherichia coli. The properties of the modified MsrB protein were compared directly with those of MsrA. Also, we have shown that in Staphylococcus aureus there are two MsrA and one nonselenoprotein MsrB, which demonstrates the same substrate stereospeci- ficity as the mouse MsrB. Key Words: methionine sulfoxide; oxidative stress; methionine sulfoxide reductase; free radicals; methio- nine oxidation; selenoprotein; stereospecificity. Methionine residues of proteins are readily oxidized to methionine sulfoxide (MetO) by most reactive oxy- gen species However, in contrast to most other oxida- tive posttranslational modifications, the oxidation of methionine residues is repaired by the action of methi- onine sulfoxide reductase (MsrA) which catalyzes re- duction of MetO to methionine, both in vitro and in vivo (1, 2). In addition to limiting the steady-state level of oxidized methionine, the cyclic oxidation/reduction of protein methionine residues constitutes a mechanism for the scavenging of ROS, and thereby provides in- creased resistance to oxidative cellular damage and to enhanced survival under conditions of oxidative stress (3–5). It was shown previously that a mouse strain lacking MsrA is more prone to oxidative stress damage, has shorter life span and exhibits a typical “tip toe” walking behavior starting at six months of age (5). Other studies have shown that the level of MsrA de- clines with age (6), and that over expression of the enzyme in human T cells increases their survival rate under conditions of oxidative stress. Furthermore, in addition to its repair and antioxidant functions, MsrA may play a regulatory role in regulation of various biological functions (7, 8). In view of the fact that the ROS-mediated oxidation of protein-bound methionine residues leads to a race- mic mixture of the R and S forms of MetO, it was disturbing that MsrA exhibits high specificity toward the S form only (9, 10). Moreover, an enzyme (Fmsr) that catalyzes specifically the reduction of free MetO, is also specific for the S isomer (9). Nevertheless, results of other studies indicated that cells have the ability to convert the R form of MetO to methionine, by an un- known mechanism (9). In an effort to identify the en- zyme(s) responsible for the conversion of R-MetO to methionine, we examined the substrate stereospecific- ity of three different Msrs in Staphylococcus aureus (11), and also of the MsrB form of enzyme found in extracts of MsrA -/- mouse (5). MATERIALS AND METHODS Methionine sulfoxide reductase activity. Enzymatic activity of methionine sulfoxide reductase was measured using either dabsyl- methionine sulfoxide or free methionine sulfoxide (R or S form) as substrate, as previously described (9). Briefly, the reaction mixture contained 1 mM substrate, 20 mM DTT, and 25 mM Tris–HCl, pH 7.4, or PBS. Following incubation for 30 min at 37°C, the reaction mixture was injected into a C18 column (Apex, Jones Chromatogra- 1 To whom correspondence and reprint requests should be ad- dressed. Fax: 301-496-0599. E-mail: moskovij@nhlbi.nih.gov. Biochemical and Biophysical Research Communications 290, 62– 65 (2002) doi:10.1006/bbrc.2001.6171, available online at http://www.idealibrary.com on 62 0006-291X/02