Thioredoxin reductase explained

Thioredoxin reductases (TR, TrxR) are enzymes that reduce thioredoxin (Trx).[1] Two classes of thioredoxin reductase have been identified: one class in bacteria and some eukaryotes and one in animals. In bacteria TrxR also catalyzes the reduction of glutaredoxin like proteins known as NrdH.[2] [3] [4] Both classes are flavoproteins which function as homodimers. Each monomer contains a FAD prosthetic group, a NADPH binding domain, and an active site containing a redox-active disulfide bond.[5]

Cellular role

Thioredoxin reductases are enzymes that catalyze the reduction of thioredoxin[1] and hence they are a central component in the thioredoxin system. Together with thioredoxin (Trx) and NADPH this system's most general description is as a system for reducing disulfide bonds in cells. Electrons are taken from NADPH via TrxR and are transferred to the active site of Trx, which goes on to reduce protein disulfides or other substrates.[6] The Trx system exists in all living cells and has an evolutionary history tied to DNA as a genetic material, defense against oxidative damage due to oxygen metabolism, and redox signaling using molecules like hydrogen peroxide and nitric oxide.[7] [8]

Diversity

Two classes of thioredoxin reductase have evolved independently:

These two classes of TrxR have only ~20% sequence identity in the section of primary sequence where they can be reliably aligned.[5] The net reaction of both classes of TrxR is identical but the mechanism of action of each is distinct.[9]

Humans express three thioredoxin reductase isozymes: thioredoxin reductase 1 (TrxR1, cytosolic), thioredoxin reductase 2 (TrxR2, mitochondrial), thioredoxin reductase 3 (TrxR3, testis specific).[10] Each isozyme is encoded by a separate gene:

thioredoxin reductase 1
Hgncid:12437
Symbol:TXNRD1
Entrezgene:7296
Omim:601112
Refseq:NM_003330
Uniprot:Q16881
Ecnumber:1.8.1.9
Chromosome:12
Arm:q
Band:23
Locussupplementarydata:-q24.1
thioredoxin reductase 2
Hgncid:18155
Symbol:TXNRD2
Entrezgene:10587
Omim:606448
Refseq:NM_006440
Uniprot:Q9NNW7
Ecnumber:1.8.1.9
Chromosome:22
Arm:q
Band:11.21
thioredoxin reductase 3
Hgncid:20667
Symbol:TXNRD3
Entrezgene:114112
Omim:606235
Refseq:XM_051264
Uniprot:Q86VQ6
Ecnumber:1.8.1.9
Chromosome:3
Arm:p
Band:13
Locussupplementarydata:-q13.33

Structure

E. coli

In E. coli ThxR there are two binding domains, one for FAD and another for NADPH. The connection between these two domains is a two-stranded anti-parallel β-sheet.[11] Each domain individually is very similar to the analogous domains in glutathione reductase, and lipoamide dehydrogenase but they relative orientation of these domains in ThxR is rotated by 66 degrees. This becomes significant in the enzyme mechanism of action which is described below.ThxR homo-dimerizes with the interface between the two monomers formed by three alpha-helices and two loops. Each monomer can separately bind a molecule of thioredoxin.

Mammalian

Mammalian TrxR structure is similar to E. coli. It contains a FAD and NADPH binding domain, and an interface between two monomer subunits. In mammalian ThxR there is an insertion in the FAD binding domain between two alpha helices which forms a small pair of beta strands.[12] The active disulfide in the enzyme is located on one of these helices and thus the active disulfide bond is located in the FAD domain and not the NADPH domain as in E. coli and other prokaryotes.

Mechanism

E. coli

In E. coli ThxR the spatial orientation of the FAD and NADPH domains are such that the redox-active rings of FAD and NADPH are not in close proximity to each other. When the FAD domain of E. coli is rotated 66 degrees with the NADPH domain remaining fixed the two prosthetic groups move into close contact allowing electrons to pass from NADPH to FAD and then to the active site disulfide bond.[13] The conserved active site residues in E. coli are -Cys-Ala-Thr-Cys-.

Mammalian

Mammalian TrxRs have a much higher sequence homology with glutathione reductase than E. coli. The active-site Cys residues in the FAD domain and bound NADPH domain are in close proximity removing the necessity for a 66 degree rotation for electron transfer found in E. coli. An additional feature of the mammalian mechanism is the presence of a selenocysteine residue at the C-terminal end of the protein which is required for catalytic activity. The conserved residues in mammalian active site are -Cys-Val-Asn-Val-Gly-Cys-.

Detection methods

Thioredoxin reductase can be quantified by various methods such as the DTNB assay using Ellman's reagent. The disulfide-based TRFS series of fluorescent probes have shown selective detection of TrxR.[14] [15] [16] [17] Mafireyi synthesized the first diselenide probe that was applied in the detection of TrxR.[18] [19] Other detection methods include immunological techniques and the selenocystine-thioredoxin reductase assay (SC-TR assay).

Clinical significance

Cancer treatment

Since the activity of this enzyme is essential for cell growth and survival, it is a good target for anti-tumor therapy. Furthermore, the enzyme is upregulated in several types of cancer, including malignant mesothelioma.[20] [21] For example, motexafin gadolinium (MGd) is a new chemotherapeutic agent that selectively targets tumor cells, leading to cell death and apoptosis via inhibition of thioredoxin reductase and ribonucleotide reductase.

Cardiomyopathy

Dilated cardiomyopathy (DCM) is a common diagnosis in cases of congestive heart failure. Thioredoxin reductases are essential proteins for regulating cellular redox balance and mitigating the damage caused by reactive oxygen species generated via oxidative phosphorylation in the mitochondria. Inactivation of mitochondrial TrxR2 in mice results in thinning of the ventricular heart walls and neonatal death. Furthermore two mutations in the TrxR2 gene are found in patients diagnosed with DCM and not in a control population. It is hypothesized that the pathological impact of these mutations is an impaired ability to control oxidative damage in cardiac myocytes.[22]

Antibiotic

There has recently been some research to show that low molecular weight thioredoxin reductase could be a target for novel antibiotics (such as auranofin or Ebselen.[23]) This is especially true for Mycobacterium Haemophilum, and could be used for antibiotic resistant bacteria.[24]

Notes and References

  1. Mustacich D, Powis G . Thioredoxin reductase . The Biochemical Journal . 346 Pt 1 . 1 . 1–8 . February 2000 . 10657232 . 1220815 . 10.1042/0264-6021:3460001 .
  2. Jordan A, Aslund F, Pontis E, Reichard P, Holmgren A . Characterization of Escherichia coli NrdH. A glutaredoxin-like protein with a thioredoxin-like activity profile . The Journal of Biological Chemistry . 272 . 29 . 18044–50 . July 1997 . 9218434 . 10.1074/jbc.272.29.18044 . free .
  3. Phulera S, Mande SC . The crystal structure of Mycobacterium tuberculosis NrdH at 0.87 Å suggests a possible mode of its activity . Biochemistry . 52 . 23 . 4056–65 . June 2013 . 23675692 . 10.1021/bi400191z .
  4. Phulera S, Akif M, Sardesai AA, Mande SC . 2014-01-01. Redox Proteins of Mycobacterium tuberculosis. Journal of the Indian Institute of Science. en. 94. 1. 127–138. 0970-4140.
  5. Hirt RP, Müller S, Embley TM, Coombs GH . The diversity and evolution of thioredoxin reductase: new perspectives . Trends in Parasitology . 18 . 7 . 302–8 . July 2002 . 12379950 . 10.1016/S1471-4922(02)02293-6 .
  6. Holmgren A, Lu J . Thioredoxin and thioredoxin reductase: current research with special reference to human disease . Biochemical and Biophysical Research Communications . 396 . 1 . 120–4 . May 2010 . 20494123 . 10.1016/j.bbrc.2010.03.083 .
  7. Meyer Y, Buchanan BB, Vignols F, Reichheld JP . Thioredoxins and glutaredoxins: unifying elements in redox biology . Annual Review of Genetics . 43 . 335–67 . 2009 . 19691428 . 10.1146/annurev-genet-102108-134201 .
  8. Lillig CH, Holmgren A . Thioredoxin and related molecules--from biology to health and disease . Antioxidants & Redox Signaling . 9 . 1 . 25–47 . Jan 2007 . 17115886 . 10.1089/ars.2007.9.25 .
  9. Arscott LD, Gromer S, Schirmer RH, Becker K, Williams CH . The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli . Proceedings of the National Academy of Sciences of the United States of America . 94 . 8 . 3621–6 . Apr 1997 . 9108027 . 20490 . 10.1073/pnas.94.8.3621 . 1997PNAS...94.3621A . free .
  10. Conrad M, Jakupoglu C, Moreno SG, Lippl S, Banjac A, Schneider M, Beck H, Hatzopoulos AK, Just U, Sinowatz F, Schmahl W, Chien KR, Wurst W, Bornkamm GW, Brielmeier M . Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function . Molecular and Cellular Biology . 24 . 21 . 9414–23 . Nov 2004 . 15485910 . 522221 . 10.1128/MCB.24.21.9414-9423.2004 .
  11. Williams CH . Mechanism and structure of thioredoxin reductase from Escherichia coli . FASEB Journal . 9 . 13 . 1267–76 . Oct 1995 . 7557016 . 10.1096/fasebj.9.13.7557016. free . 2027.42/154540 . 26055087 . free .
  12. Sandalova T, Zhong L, Lindqvist Y, Holmgren A, Schneider G . Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme . Proceedings of the National Academy of Sciences of the United States of America . 98 . 17 . 9533–8 . Aug 2001 . 11481439 . 55487 . 10.1073/pnas.171178698 . 2001PNAS...98.9533S . free .
  13. Lennon BW, Williams CH . Reductive half-reaction of thioredoxin reductase from Escherichia coli . Biochemistry . 36 . 31 . 9464–77 . Aug 1997 . 9235991 . 10.1021/bi970307j .
  14. Li X, Zhang B, Yan C, Li J, Wang S, Wei X, Jiang X, Zhou P, Fang J . 6 . A fast and specific fluorescent probe for thioredoxin reductase that works via disulphide bond cleavage . Nature Communications . 10 . 1 . 2745 . June 2019 . 31227705 . 10.1038/s41467-019-10807-8 . 6588570 . 2019NatCo..10.2745L . free .
  15. Ma H, Zhang J, Zhang Z, Liu Y, Fang J . A fast response and red emission probe for mammalian thioredoxin reductase . Chemical Communications . 52 . 81 . 12060–12063 . October 2016 . 27709154 . 10.1039/C6CC04984B .
  16. Zhao J, Qu Y, Gao H, Zhong M, Li X, Zhang F, Chen Y, Gan L, Hu G, Zhang H, Zhang S, Fang J . 6 . Loss of thioredoxin reductase function in a mouse stroke model disclosed by a two-photon fluorescent probe . Chemical Communications . 56 . 90 . 14075–14078 . November 2020 . 33107534 . 10.1039/D0CC05900E . 225082279 .
  17. Liu Y, Ma H, Zhang L, Cui Y, Liu X, Fang J . A small molecule probe reveals declined mitochondrial thioredoxin reductase activity in a Parkinson's disease model . Chemical Communications . 52 . 11 . 2296–9 . February 2016 . 26725656 . 10.1039/c5cc09998f .
  18. Mafireyi TJ, Laws M, Bassett JW, Cassidy PB, Escobedo JO, Strongin RM . A Diselenide Turn-On Fluorescent Probe for the Detection of Thioredoxin Reductase . Angewandte Chemie . 59 . 35 . 15147–15151 . August 2020 . 32449244 . 10.1002/ange.202004094 . 9438933 . 2020AngCh.13215259M . 229142596 .
  19. Mafireyi TJ, Escobedo JO, Strongin RM . 2021-03-29. Fluorogenic probes for thioredoxin reductase activity . Results in Chemistry. 3. en. 100127. 10.1016/j.rechem.2021.100127. 2211-7156. free.
  20. Nilsonne G, Sun X, Nyström C, Rundlöf AK, Potamitou Fernandes A, Björnstedt M, Dobra K . Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress . Free Radical Biology & Medicine . 41 . 6 . 874–85 . Sep 2006 . 16934670 . 10.1016/j.freeradbiomed.2006.04.031 . 10616/47514 . free .
  21. Kahlos K, Soini Y, Säily M, Koistinen P, Kakko S, Pääkkö P, Holmgren A, Kinnula VL . Up-regulation of thioredoxin and thioredoxin reductase in human malignant pleural mesothelioma . International Journal of Cancer . 95 . 3 . 198–204 . May 2001 . 11307155 . 10.1002/1097-0215(20010520)95:3<198::AID-IJC1034>3.0.CO;2-F . free .
  22. Sibbing D, Pfeufer A, Perisic T, Mannes AM, Fritz-Wolf K, Unwin S, Sinner MF, Gieger C, Gloeckner CJ, Wichmann HE, Kremmer E, Schäfer Z, Walch A, Hinterseer M, Näbauer M, Kääb S, Kastrati A, Schömig A, Meitinger T, Bornkamm GW, Conrad M, von Beckerath N . Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 cause dilated cardiomyopathy . European Heart Journal . 32 . 9 . 1121–33 . May 2011 . 21247928 . 10.1093/eurheartj/ehq507 . free . 11858/00-001M-0000-0024-1F10-3 . free .
  23. Marshall AC, Kidd SE, Lamont-Friedrich SJ, Arentz G, Hoffmann P, Coad BR, Bruning JB . Aspergillus fumigatus Thioredoxin Reductase . Antimicrobial Agents and Chemotherapy . 63 . 3 . March 2019 . 30642940 . 6395915 . 10.1128/AAC.02281-18 .
  24. Harbut MB, Vilchèze C, Luo X, Hensler ME, Guo H, Yang B, Chatterjee AK, Nizet V, Jacobs WR, Schultz PG, Wang F . 6 . Auranofin exerts broad-spectrum bactericidal activities by targeting thiol-redox homeostasis . Proceedings of the National Academy of Sciences of the United States of America . 112 . 14 . 4453–8 . April 2015 . 25831516 . 4394260 . 10.1073/pnas.1504022112 . 2015PNAS..112.4453H . free .

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