title: Tandem use of selenocysteine: adaptation of a selenoprotein glutaredoxin for reduction of selenoprotein methionine sulfoxide reductase.
abstract: Several engineered selenocysteine (Sec)-containing glutaredoxins (Grxs) and their enzymatic properties have been reported, but natural selenoprotein Grxs have not been previously characterized. We expressed a bacterial selenoprotein Grx from Clostridium sp. (also known as Alkaliphilus oremlandii) OhILAs in Escherichia coli and characterized this selenoenzyme and its natural Cys homologues in Clostridium and E. coli. The selenoprotein Grx had a 200-fold higher activity than its Sec-to-Cys mutant form, suggesting that Sec is essential for catalysis by this thiol-disulfide oxidoreductase. Kinetic analysis also showed that the selenoprotein Grx had a 10-fold lower K(m) than Cys homologues. Interestingly, this selenoenzyme efficiently reduced a Clostridium selenoprotein methionine sulfoxide reductase A (MsrA), suggesting that it is the natural reductant for the protein that is not reducible by thioredoxin, a common reductant for Cys-containing MsrAs. We also found that the selenoprotein Grx could not efficiently reduce a Cys version of Clostridium MsrA, whereas natural Clostridium and E. coli Cys-containing Grxs, which efficiently reduce Cys-containing MsrAs, poorly acted on the selenoprotein MsrA. This specificity for MsrA reduction could explain why Sec is utilized in Clostridium Grx and more generally provides a novel example of the use of Sec in biological systems.
authors.affiliation: Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Korea.
authors.forename: Moon-Jung
authors.initials: MJ
authors.lastname: Kim
authors.forename: Byung Cheon
authors.initials: BC
authors.lastname: Lee
authors.forename: Jaeho
authors.initials: J
authors.lastname: Jeong
authors.forename: Kong-Joo
authors.initials: KJ
authors.lastname: Lee
authors.forename: Kwang Yeon
authors.initials: KY
authors.lastname: Hwang
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Gladyshev
authors.forename: Hwa-Young
authors.initials: HY
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003013:Clostridium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D054477:Glutaredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051150:Methionine Sulfoxide Reductases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017279:Selenocysteine / Q000378:metabolism; D051140:Selenoproteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D054477:Glutaredoxins; D051140:Selenoproteins; D017279:Selenocysteine; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase