Selenium in mammalian thioredoxin reductase

Abstract

Thioredoxin reductase (TrxR) is a major enzyme in regulating intracellular redox status. The enzyme from several mammalian tissues has been purified, showing higher molecular weight and a much broader substrate specificity than E. coli TrxR, but the structural basis of these properties remained unknown.

We have cloned a cDNA of rat TrxR and sequenced the major part of peptides derived from bovine TrxR. The primary structure was surprisingly not similar to E. coli TrxR but homologous to glutathione reductase (GR) with an identical redox-active dithiol motif but a unique C-terminal elongation, which contains a selenocysteine (SeCys) residue in the conserved sequence -Gly-Cys-SeCys-Gly. We also identified that in the 3'-untranslated region of the rat mRNA, there was a SeCys insertion sequence (SECIS) motif that is required for translation of the UGA "stop" codon as selenocysteine.

Our initial attempts to express the rat TrxR gene in E. coli were unsuccessful, since the mechanism used for translation of UGA as SeCys is not conserved between E. coli and mammalian cells. We therefore changed the SeCys codon to Cys or Ser codon, as well as removed the SECIS motif. The truncated protein having the C-terminal SeCys-Gly dipeptide deleted was also engineered. All three mutant enzymes were expressed in E. coli and purified to homogeneity as flavoproteins. In anaerobic titrations with NADPH, all three mutant enzymes rapidly generated the absorbance centered at 540 nm, a common characteristic of mammalian TrxR. This result was consistent with an intact reductive-half reaction involving transfer of electrons from NADPH to the redox-active disulfide via FAD, and ruled out a role for the SeCys-Gly dipeptide in FAD binding. However, only the SeCys498Cys TrxR showed catalytic activity in reducing thioredoxin, with a 100-fold lower kcat and a 10-fold lower Km compared to the wild-type rat TrxR. The pH optimum of the SeCys498Cys TrxR was 9 as opposed to 7 for the wild-type TrxR, strongly suggesting involvement of the low pKa SeCys selenol in the enzyme catalytic mechanism. Whereas H202 is a substrate for the wild-type enzyme, all mutant enzymes lack the activity in reducing H2O2. Thus, selenium is required for the catalytic activities of mammalian TrxR.

By chemical modification and peptide sequencing analyses of the oxidized enzyme, we demonstrated two redox-active bridges: one was a disulfide with the sequence -Cys59 -Val-Asn-Val-Gly-Cys64-, identical to the active site of GR; the other was a selenenyl-sulfide formed from Cys497-SeCys498 and confirmed by mass spectrometry. Both the bridges were reducible by NADPH. Based on the structure of GR, we proposed that in TrxR the C-terminal CYS497-SeCys498 residues of one subunit were adjacent to the CYS59 and Cys64 residues of the second subunit. The reductive half-reaction of TrxR includes electron transfer from the nascent CYS59 and Cys64 dithiol of one subunit to the selenenylsulfide of the other subunit to generate the active site selenolthiol. We have also postulated mechanisms of mammalian TrxR in reduction of thioredoxin and H2O2.

We have crystallized the rat SeCys498Cys mutant TrxR. X-ray diffraction pattern extends to 3 Å resolution, which shows that monoclinic crystals have unit cell dimensions of a = 79.9 Å, b = 140.5 Å, c = 170.8 Å and ß = 94.6 Å. Analysis of the three-dimensional structure is in progress to understand the structure-function relationship of mammalian TrxR with its unique requirement for selenium.