The Arabidopsis Plastidic Methionine Sulfoxide Reductase B Proteins. Sequence and Activity Characteristics, Comparison of the Expression with Plastidic Methionine Sulfoxide Reductase A, and Induction by Photooxidative Stress

Abstract

Two types of methionine (Met) sulfoxide reductases (Msr) catalyze the reduction of Met sulfoxide (MetSO) back to Met. MsrA, well characterized in plants, exhibits an activity restricted to the Met-S-SO-enantiomer. Recently, a new type of Msr enzyme, called MsrB, has been identified in various organisms and shown to catalytically reduce the R-enantiomer of MetSO. In plants, very little information is available about MsrB and we focused our attention on Arabidopsis (Arabidopsis thaliana) MsrB proteins. Searching Arabidopsis genome databases, we have identified nine open reading frames encoding proteins closely related to MsrB proteins from bacteria and animal cells. We then analyzed the activity and abundance of the two chloroplastic MsrB proteins, MsrB1 and MsrB2. Both enzymes exhibit an absolute R-stereospecificity for MetSO and a higher catalytic efficiency when using protein-bound MetSO as a substrate than when using free MetSO. Interestingly, we observed that MsrB2 is reduced by thioredoxin, whereas MsrB1 is not. This feature of MsrB1 could result from the lack of the catalytical cysteine (Cys) corresponding to Cys-63 in Escherichia coli MsrB that is involved in the regeneration of Cys-117 through the formation of an intramolecular disulfide bridge followed by thioredoxin reduction. We investigated the abundance of plastidial MsrA and B in response to abiotic (water stress, photooxidative treatment) and biotic (rust fungus) stresses and we observed that MsrA and B protein levels increase in response to the photooxidative treatment. The possible role of plastidic MsrB in the tolerance to oxidative damage is discussed.Oxygen is essential to all aerobic organisms but can also lead to many harmful effects (Davies, 1995). Proteins are easily damaged by reactive oxygen species, with Met being one of the amino acid residues most susceptible to oxidation (Dann and Pell, 1989). The generation of Met sulfoxide (MetSO) is mediated by various biological oxidants such as hydrogen peroxide, hydroxyl radicals, ozone as well as by metals, and results in modifications of activity and conformation for many proteins (Gao et al., 1998;Davis et al., 2000). Oxidation of Met residues is readily reversed by the action of an enzyme initially referred as peptide MetSO reductase (PMSR), which catalyzes the thioredoxin-dependent reduction of MetSO back to Met (Brot et al., 1981). The enzyme is present in most living organisms and has been described as belonging to the minimal set of proteins sufficient for cell life (Mushegian and Koonin, 1996). PMSR has a protective role against oxidative damage (Moskovitz et al., 1997).Indeed, pmsr null mutants of Escherichia coli and Saccharomyces cerevisiae show a decreased resistance toward oxidative stress conditions (Moskovitz et al., 1995(Moskovitz et al., , 1997. Accordingly, PMSR overexpression in S. cerevisiae or in human T-cells results in an increased resistance to oxidative treatments (Moskovitz et al., 1998). The first PMSR has been renamed Met sulfoxide reductase A (MsrA) and has been sh...

Characterization of a novel methionine sulfoxide reductase A from tomato (Solanum lycopersicum), and its protecting role in Escherichia coli

“…In this study, we found that SlMSRA2 exhibited activity with both free and protein-bound MetSO in the presence of DTT. These findings are in agreement with previous reports, which also demonstrated that MSRA was active in DTT (or DTE) systems (13,14). Moreover, the reduction of MetSO to Met was found to be DTT-dependent and its substrate was targeted to the methionine-S-sulfoxide (but not methionine-R-sulfoxide) stereoisomer.…”

“…The concentration of the dabsyl-Met was determined using a standard that was purchased from the TCI-GR Company (TCI-GR, Japan). RP-HPLC was performed as described by Vieira Dos Santos et al with minor modifications (13). Briefly, solvent A (acetate buffer 29 mM, pH 4.16) and solvent B (acetonitrile) were used in the HPLC assay.…”

Characterization of Nda2, a Plastoquinone-reducing Type II NAD(P)H Dehydrogenase in Chlamydomonas Chloroplasts

“…in buffer A (25 mM imidazole pH 7.5, 20 mM amine-triethanol, 500 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine). The supernatant was recovered by centrifugation (12,000 ϫ g for 20 min) and used for further purification of His-tagged Nda2 by metal-chelating chromatography as previously described (24). After addition of glycerol (50% v/v), the purified enzyme was stored at Ϫ20°C.…”

Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid

“…For instance, mammalian Trxs are able to covalently interact with substrateoxidized 1-Cys MSRBs and to regenerate their activity (24). In the case of plant 1-Cys MSRBs, there is generally only one isoform, termed MSRB1, which is located in plastids (20,25). Arabidopsis MSRB1 is efficiently reduced, as mentioned above, by Grxs (16,21) but also by a specific plant Trx termed CDSP32 (Chloroplastic Drought-induced Stress Protein of 32 kDa) (16).…”