Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue.
We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.
Zhong
L
L
Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Arnér
E S
ES
Ljung
J
J
Aslund
F
F
Holmgren
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000737:chemistry; D005980:Glutathione Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011725:Pyridines; D051381:Rats; D012643:Selenium / Q000032:analysis; D017279:Selenocysteine; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology
D018076:DNA, Complementary; D010446:Peptide Fragments; D011725:Pyridines; D017279:Selenocysteine; D009249:NADP; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D012643:Selenium; C029351:4-vinylpyridine