Abstract
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.
Highlights
Thioredoxin reductase is a dimeric enzyme with a redoxactive disulfide and an FAD in each monomer, and it is a member of a larger family of pyridine nucleotide-disulfide oxidoreductases, which includes the closely related enzymes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, and mercuric ion reductase [1]
Since we found that human and calf Thioredoxin reductase (TrxR) contained the same amount of selenium, we continued our ongoing study of the calf enzyme, with a special emphasis on the structure of the enzyme and the significance of the selenium content for its catalytic properties
We found that a selenocysteine containing COOH-terminal motif (-Gly-Cys-SeCys-Gly-COOH) is conserved among rat, calf, and human TrxR and that both rat and human cDNA sequences carry consensus sequences in the 3ЈUTR necessary for the proper incorporation of selenocysteine during translation of the mRNA
Summary
A peptide sequence from the protein purified from the human T cell line [24] agreed with a putative human placental TrxR cDNA sequence showing homology to glutathione reductase but not prokaryotic TrxR [26]. The putative human placental cDNA clone did not give any active enzyme, since expression in E. coli resulted in a protein of the correct Mr, which, did not incorporate FAD [26]. Together with the sequence of the translated open reading frame of a rat TrxR cDNA clone, the close structural homology to glutathione reductase was evident, and the earlier published putative human cDNA clone could be confirmed. We found that a carboxyl-terminal motif containing cysteine and selenocysteine was conserved among human, rat, and bovine TrxR, and we present evidence that the carboxyl terminus is essential for the catalytic activity of the enzyme. Preliminary results have been reported [27]
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