Mouse methionine sulfoxide reductase B: effect of selenocysteine incorporation on its activity and expression of the seleno-containing enzyme in bacterial and mammalian cells.
The mammalian methionine sulfoxide reductase B (MsrB) has been found to be a selenoprotein which can reduce R form of both free and protein-incorporated methionine sulfoxide to methionine. Together with MsrA, which reduces specifically the S form of methionine sulfoxide, the living cell can repair methionine-damaged proteins and salvage free methionine under oxidative stress conditions. Here, we report about the pivotal role of the selenocysteine residue in the protein putative active site by site-directed mutagenesis directed to the selenocysteine codon. Using the Escherichia coli SECIS (selenocysteine insertion sequence) element, needed for the recognition of the UGA codon as a selenocysteine codon in E. coli, we expressed the seleno-MsrB as a recombinant selenoprotein in E. coli. The recombinant seleno-MsrB has been shown to be much more active than the cysteine mutant, whereas the mutations to alanine and serine rendered the protein inactive. Although the yields of expression of the full-length N-terminus and C-terminus His-tagged seleno-MsrB were only 3% (of the total MsrB expressed), the C-terminus His-tagged protein enabled us to get a pure preparation of the seleno-MsrB. Using both recombinant selenoproteins, the N-terminus His-tagged and the C-terminus His-tagged proteins, we were able to determine the specific activities of the recombinant seleno-MsrB, which were found to be much higher than the cysteine mutant homologue. This finding confirmed our suggestion that the selenocysteine is essential for maintaining high reducing activity of MsrB. In addition, using radioactive selenium we were able to determine the in vivo presence of MsrB as a selenoprotein in mammalian cell cultures.
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002460:Cell Line; D003553:Cystine / Q000031:analogs & derivatives* / Q000378:metabolism*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051150:Methionine Sulfoxide Reductases; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016566:Organoselenium Compounds / Q000378:metabolism*; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D051140:Selenoproteins; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D016566:Organoselenium Compounds; D011506:Proteins; D011994:Recombinant Proteins; D051140:Selenoproteins; C009226:selenocystine; D003553:Cystine; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase