NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Thioredoxin Reductases

Thioredoxin reductases (TRs) are oxidoreductases that comprise the major disulfide reduction system of the cell by providing of redox equivalent to thioredoxins [ 1, 2, 3 ]. Thioredoxin reductases are NADPH-dependent ezymes that belong to pyridine nucleotide disulfide oxidoreductase family [ 1, 4 ].

The mechanism of protein disulfide reduction catalized by thioredoxin system.

There are two forms of TR [ 1, 4, 5 ]:

1) Bacterial-type TR is a homodimer of ~35-kD subunits and is present in bacteria, archaea, and lower eukaryotes, including plants and fungi. This enzyme uses a CxxC motif for substrate reduction, accept electrons from a protein-bound flavin adenine dinucleotide (FAD) and further transfer them to oxidized thioredoxin using extensive conformational changes and domain rearrangements. The bacterial-type TR is also called a small TR, which distinguishes this enzyme from the second TR form, designated large TR.

2) Large TRs are highly homologous to glutathione reductases, trypanothione reductases, and lipoamide dehydrogenases and have no sequence similarity to small TRs. Active sites of large TRs as well as of their close homologs use a CxxxxC motif (two cysteines separated by four other residues) that forms a reversible disulfide bond during the catalytic cycle. Large TRs had been found only in animals and are often called animal TRs. Most of these enzymes contain a selenocysteine (Sec) residue present in a carboxy-terminal Gly-Cys-Sec-Gly motif, which can reduce thioredoxins [ 6 ]. Because of the presence of Sec, animal TRs have broad substrate specificity and can reduce a variety of proteins and low molecular weight compounds. [ 1, 2, 3 ]. Some members of the large TR family, such as two Drosophila TRs and one of two Caenorhabditis elegans TRs have cysteine in place of Sec. [ 7, 8 ].

3D structure of E. coli Tioredoxin reductase, Rat Tioredoxin reductase 1 and Mouse Thioredoxin Reductase 3.


There are 177 solved Tioredoxin Reductase structures in Protein Data Bank at this time (June 2015).

There are three TR paralogs in mammalian cells. All of them are selenocysteine-containing proteins and Sec residue is located in the C-terminal penultimate position [ 1, 5, 9 ]. Thioredoxin reductase 1 (TR1) is primarily localized in the cytosol and nucleus. Although cytosolic thioredoxin (Trx1) serves as the major substrate for TR1, TR1 can also reduce a variety of low-molecular-weight compounds [ 1, 2 ]. There are at least six different TR1 isoforms in mammals, which are produced by alternative splicing and N-terminal extension of the protein due to use of distinct transcription initiation sites [ 1, 10, 11 ]. The second member of the TR protein family is thioredoxin reductase 3 (TR3). TR3 is localized in the mitochondria, where it is involved in reduction of mitochondrial thioredoxin (Trx2) and glutaredoxin 2 (Grx2) [ 11 ]. Similar to TR1, multiple TR3 isoforms are known. Both TR1 and TR3 are present in all vertebrates, and knockout of either of them leads to embryonic lethality in mice. [ 13, 14, 15 ]. The third TR homolog in mammals is thioredoxin/glutathione reductase (TR2 or TGR). This protein contains an additional glutaredoxin (Grx) domain, which is located in the N-terminal part of the protein [ 16 ]. Due to the presence of the Grx domain, TGR displays Grx activity, suggesting that this protein is involved in both Trx and GSH systems. Physiological function of TGR remains unknown. TGR is expressed at high levels in testis after puberty, and it was proposed that TGR might be involved in the formation/isomerization of disulfide bonds during sperm maturation [ 17 ]. Two distinct TGR isoforms are found in mice. The long form is produced by an alternative CUG start codon, as opposed to the normally used AUG codon [ 18 ].

Domain organization for mammalian TR1 and TR2 (TGR).


Yeast Saccharomyces Cerevisiae Thioredoxin Reductases (small or "bacterial" type of TRs):

>gi|6320560|ref|NP_010640.1| Trr1 [Saccharomyces cerevisiae]
MVHNKVTIIGSGPAAHTAAIYLARAEIKPILYEGMMANGIAAGGQLTTTTEIENFPGFPDGLTGSELMDRMREQSTKFGTEIITETVSKVDLSSKPFKLWTEFNEDAE
PVTTDAIILATGASAKRMHLPGEETYWQKGISACAVCDGAVPIFRNKPLAVIGGGDSACEEAQFLTKYGSKVFMLVRKDHLRASTIMQKRAEKNEKIEILYNTVALEA
KGDGKLLNALRIKNTKKNEETDLPVSGLFYAIGHTPATKIVAGQVDTDEAGYIKTVPGSSLTSVPGFFAAGDVQDSKYRQAITSAGSGCMAALDAEKYLTSLE

>gi|6321898|ref|NP_011974.1| Trr2 [Saccharomyces cerevisiae]
MIKHIVSPFRTNFVGISKSVLSRMIHHKVTIIGSGPAAHTAAIYLARAEMKPTLYEGMMANGIAAGGQLTTTTDIENFPGFPESLSGSELMERMRKQSAKFGTNIITE
TVSKVDLSSKPFRLWTEFNEDAEPVTTDAIILATGASAKRMHLPGEETYWQQGISACAVCDGAVPIFRNKPLAVIGGGDSACEEAEFLTKYASKVYILVRKDHFRASV
IMQRRIEKNPNIIVLFNTVALEAKGDGKLLNMLRIKNTKSNVENDLEVNGLFYAIGHSPATDIVKGQVDEEETGYIKTVPGSSLTSVPGFFAAGDVQDSRYRQAVTSA
GSGCIAALDAERYLSAQE


Human Thioredoxin Reductases (large or "animal" type of TRs):

>gi|148277071|ref|NP_001087240.1| thioredoxin reductase 1 (TR1), cytoplasmic isoform 3 [Homo sapiens]
MGCAEGKAVAAAAPTELQTKGKNGDGRRRSAKDHHPGKTLPENPAGFTSTATADSRALLQAYIDGHSVVIFSRSTCTRCTEVKKLFKSLCVPYFVLELDQTEDGRALE
GTLSELAAETDLPVVFVKQRKIGGHGPTLKAYQEGRLQKLLKMNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIP
KKLMHQAALLGQALQDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIATGERPRYLGIPGDK
EYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEG
EYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKA
VEKFGEENIEVYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFTTLSVTKRSGASILQAGCU
G

>gi|291045266|ref|NP_443115.1| thioredoxin reductase 2 (TGR or TR2) isoform 1 [Homo sapiens]
MERSPPQSPGPGKAGDAPNRRSGHVRGARVLSPPGRRARLSSPGPSRSSEAREELRRHLVGLIERSRVVIFSKSYCPHSTRVKELFSSLGVECNVLELDQVDDGARVQ
EVLSEITNQKTVPNIFVNKVHVGGCDQTFQAYQSGLLQKLLQEDLAYDYDLIIIGGGSGGLSCAKEAAILGKKVMVLDFVVPSPQGTSWGLGGTCVNVGCIPKKLMHQ
AALLGQALCDSRKFGWEYNQQVRHNWETMTKAIQNHISSLNWGYRLSLREKAVAYVNSYGEFVEHHKIKATNKKGQETYYTAAQFVIATGERPRYLGIQGDKEYCITS
DDLFSLPYCPGKTLVVGASYVALECAGFLAGFGLDVTVMVRSILLRGFDQEMAEKVGSYMEQHGVKFLRKFIPVMVQQLEKGSPGKLKVLAKSTEGTETIEGVYNTVL
LAIGRDSCTRKIGLEKIGVKINEKSGKIPVNDVEQTNVPYVYAVGDILEDKPELTPVAIQSGKLLAQRLFGASLEKCDYINVPTTVFTPLEYGCCGLSEEKAIEVYKK
ENLEIYHTLFWPLEWTVAGRENNTCYAKIICNKFDHDRVIGFHILGPNAGEVTQGFAAAMKCGLTKQLLDDTIGIHPTCGEVFTTLEITKSSGLDITQKGCUG

>gi|22035672|ref|NP_006431.2| thioredoxin reductase 3 (TR3), mitochondrial isoform 1 [Homo sapiens]
MAAMAVALRGLGGRFRWRTQAVAGGVRGAARGAAAGQRDYDLLVVGGGSGGLACAKEAAQLGRKVAVVDYVEPSPQGTRWGLGGTCVNVGCIPKKLMHQAALLGGLIQ
DAPNYGWEVAQPVPHDWRKMAEAVQNHVKSLNWGHRVQLQDRKVKYFNIKASFVDEHTVCGVAKGGKEILLSADHIIIATGGRPRYPTHIEGALEYGITSDDIFWLKE
SPGKTLVVGASYVALECAGFLTGIGLDTTIMMRSIPLRGFDQQMSSMVIEHMASHGTRFLRGCAPSRVRRLPDGQLQVTWEDSTTGKEDTGTFDTVLWAIGRVPDTRS
LNLEKAGVDTSPDTQKILVDSREATSVPHIYAIGDVVEGRPELTPIAIMAGRLLVQRLFGGSSDLMDYDNVPTTVFTPLEYGCVGLSEEEAVARHGQEHVEVYHAHYK
PLEFTVAGRDASQCYVKMVCLREPPQLVLGLHFLGPNAGEVTQGFALGIKCGASYAQVMRTVGIHPTCSEEVVKLRISKRSGLDPTVTGCUG


The major assays for thioredoxin reductase activity measurement [ 19 ].


A) Spectrophotometric assay of insulin reduction: Final oxidation of NADPH is followed as the decrease of absorbance at 340 nm [ 19 ].

1) Trx-S2 + TRred → Trx-(SH)2 + TRox ;

2) Trx-(SH)2 + Insulin-S2 → Trx-S2 + Insulin-(SH)2 ;

3) TRox + NADPH → TRred + NADP+ ;

B) Spectrophotometric assay using 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB) as substrate: TR uses NADPH to reduce DTNB to 5-thio-2-nitrobenzoic acid (TNB). TR activity is determined by the increase of absorbance at 412 nm. Using of TR inhibitors allows correction of non-thioredoxin reductase-independent DTNB reduction [ 19, 20, 21 ].

DTNB + NADPH + TR + H+ NADPH ⇄ 2TNB + NADP+ ;


There are lot of commertially avaliable thioredoxin reductase assay kits. Most of them are based on DTNB reduction. Fluorescently labeled substrate, such as di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), can be used for detection [ 19 ].

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