Selenium utilization in thioredoxin and catalytic advantage provided by selenocysteine.
Thioredoxin (Trx) is a major thiol-disulfide reductase that plays a role in many biological processes, including DNA replication and redox signaling. Although selenocysteine (Sec)-containing Trxs have been identified in certain bacteria, their enzymatic properties have not been characterized. In this study, we expressed a selenoprotein Trx from Treponema denticola, an oral spirochete, in Escherichia coli and characterized this selenoenzyme and its natural cysteine (Cys) homologue using E. coli Trx1 as a positive control. (75)Se metabolic labeling and mutation analyses showed that the SECIS (Sec insertion sequence) of T. denticola selenoprotein Trx is functional in the E. coli Sec insertion system with specific selenium incorporation into the Sec residue. The selenoprotein Trx exhibited approximately 10-fold higher catalytic activity than the Sec-to-Cys version and natural Cys homologue and E. coli Trx1, suggesting that Sec confers higher catalytic activity on this thiol-disulfide reductase. Kinetic analysis also showed that the selenoprotein Trx had a 30-fold higher Km than Cys-containing homologues, suggesting that this selenoenzyme is adapted to work efficiently with high concentrations of substrate. Collectively, the results of this study support the hypothesis that selenium utilization in oxidoreductase systems is primarily due to the catalytic advantage provided by the rare amino acid, Sec.
Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea.
Moon-Jung
MJ
Kim
Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Byung Cheon
BC
Lee
Division of Biotechnology, College of Life Sciences & Biotechnology, Korea University, Seoul 136-701, Republic of Korea.
Kwang Yeon
KY
Hwang
Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Vadim N
VN
Gladyshev
Department of Biochemistry and Molecular Biology, Yeungnam University College of Medicine, Daegu 705-717, Republic of Korea. Electronic address: hykim@ynu.ac.kr.
Hwa-Young
HY
Kim
D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D011485:Protein Binding; D012643:Selenium / Q000737:chemistry*; D017279:Selenocysteine / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013879:Thioredoxins / Q000737:chemistry*; D048373:Treponema denticola / Q000201:enzymology*
D017279:Selenocysteine; D013879:Thioredoxins; D012643:Selenium
Disulfide reductase; Selenoprotein; Thioredoxin; Treponema denticola