Mitochondrial thioredoxin reductase in bovine adrenal cortex

Abstract

Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH-dependent reduction of 5,5 H dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, dl-a-lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment. The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.

Structure and mechanism of mammalian thioredoxin reductase: The active site is a redox-active selenolthiol/selenenylsulfide formed from the conserved cysteine-selenocysteine sequence

“…All TrxR from mammalian cells, of which there are a growing number of isoenzymes (19,(31)(32)(33), have the conserved C-terminal sequence -Gly-Cys-SeCys-Gly and a subunit with homology to GR (10) including the identical conserved active site of the latter enzyme (19,(31)(32)(33). However, the detailed mechanism of mammalian TrxR has not been known.…”

Selenium in Biology: Facts and Medical Perspectives

“…By reduction of hydroperoxides to the corresponding alcohols, these enzymes can prevent the production of reactive oxygen radicals and thus may contribute to the protection of the Selenium in Biology 851 Ursini et al, 1982;Brigelius-Flohé et al, 1994 Plasma GPx (pGPx, GPx-3) Takahashi et al, 1987 Gastrointestinal GPx (GI-GPx, GPx-GI, GPx-2) Chu et al, 1993 Iodothyronine deiodinases 5Ј-deiodinase, type I (5ЈDI) Behne et al, 1990;Arthur et al, 1990 5Ј-deiodinase Lee et al, 1999;Watabe et al, 1999;Miranda-Vizuete et al, 1999;Gasdaska et al, 1999 Thioredoxin reductase homologs (SelZf1; SelZf2) Lescure et al, 1999 Selenophosphate synthetase-2 Guimaraes et al, 1996 Functionally undefined 15 kDa selenoprotein of T cells Gladyshev et al, 1998 Selenoprotein P 10 (SelP) Motsenbocker and Tappel organism's macromolecules and biomembranes against oxidation (Sies et al, 1972;Flohé, 1989;Ursini et al, 1995). The role of the cytoplasmic GPx as an 'emergency enzyme' to fight oxidative stress was verified by reverse genetics (Ho et al, 1997;Cheng et al, 1998;de Haan et al, 1998;Fu et al, 1999;Jaeschke et al, 1999) and, in this role, cGPx cannot be substituted by any of the other selenoproteins.…”

“…It needs selenocysteine as the penultimate amino acid residue for its appropriate enzymatic function (Marcocci et al, 1997;Gromer et al, 1998;Lee et al, 2000;Gorlatov and Stadtman, 2000). Recently, two more tissue-specifically expressed isoenzymes were also identified as selenoproteins (Gasdaska et al, 1999;Lee et al, 1999;Watabe et al, 1999) and related proteins were identified by 'in silico' cloning (Lescure et al, 1999). Various natural and synthetic compounds, apart from disulfide groups in peptides and proteins, can be reduced by TrxR Björnstedt et al, 1995).…”

Heterogeneity within Animal Thioredoxin Reductases

“…Disruption of the gene for thioredoxin 1, a 12-kDa thiol disulfide oxidoreductase, results in embryonic lethality in mice (4) demonstrating an essential role of the thioredoxin system in the development of mammals. Three distinct thioredoxin reductases (TRs) 1 (5)(6)(7)(8)(9)(10) are responsible for maintaining thioredoxins in a reduced state and are also capable of reducing a great variety of other protein and nonprotein redox substrates. The cytosolic thioredoxin reductase (TR1), the most characterized of the three enzymes, was known for decades, but only recently it was found to be a selenium-containing protein (11).…”

“…Recently, two additional thioredoxin reductases, TR2 and TR3, were identified that contained the conserved selenocysteine residue (5)(6)(7)(8)(9)(10). These enzymes had also other sequences essential for catalytic activity, including an N-terminal disulfide active center, NADPH-and FAD-binding domains and dimer interface sequences (5)(6)(7)(8)(9)(10).…”