Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans.
Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.
Lee
Byung Cheon
BC
College of Life and Environmental Sciences, Korea University, Seoul 136-701, Republic of Korea.
Lee
Yong Kwon
YK
Lee
Ho-Joung
HJ
Stadtman
Earl R
ER
Lee
Kweon-Haeng
KH
Chung
Namhyun
N
D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017173:Caenorhabditis elegans; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D051150:Methionine Sulfoxide Reductases; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013696:Temperature
D000975:Antioxidants; D018076:DNA, Complementary; D011994:Recombinant Proteins; D009249:NADP; D004228:Dithionitrobenzoic Acid; D008715:Methionine; D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase; D004229:Dithiothreitol