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Methionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and nucleus and exhibited the highest methionine-R-sulfoxide reductase activity because of the presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs contained cysteine in place of Sec and were less catalytically efficient. MsrB2 (CBS-1) resided in mitochondria. It had high affinity for methionine-R-sulfoxide, but was inhibited by higher concentrations of the substrate. The human MsrB3 gene gave rise to two protein forms, MsrB3A and MsrB3B. These were generated by alternative splicing that introduced contrasting N-terminal and C-terminal signals, such that MsrB3A was targeted to the endoplasmic reticulum and MsrB3B to mitochondria. We found that only mitochondrial forms of mammalian MsrBs (MsrB2 and MsrB3B) could compensate for MsrA and MsrB deficiency in yeast. All mammalian MsrBs belonged to a group of zinc-containing proteins. The multiplicity of MsrBs contrasted with the presence of a single mammalian MsrA gene as well as with the occurrence of single MsrA and MsrB genes in yeast, fruit flies, and nematodes. The data suggested that different cellular compartments in mammals maintain a system for repair of oxidized methionine residues and that this function is tuned in enzyme- and stereo-specific manner.