The Mechanism of High MR Thioredoxin Reductase Investigated by Semisynthesis and Crystallography

The high Mr (~55 kDa) thioredoxin reductases (TR) characteristic of higher eukaryotes are members of the glutathione reductase (GR) family of pyridine nucleotide disulfide oxidoreductases. These homodimeric enzymes catalyze the reduction of a cognate disulfide substrate. During the enzymatic cycle, reducing equivalents pass from NADPH to the conserved active site disulfide via an enzyme-bound FAD and then to the cognate substrate. TRs are unique in the family as electrons are then transferred to the C-terminal active site of the adjacent molecule as part of a 16 amino acid extension (in place of the cognate GR substrate GSSG), prior to transfer to the substrate thioredoxin. Each electron transfer step occurs via thiol-disulfide exchange in a multi-step process mediated by a conserved catalytic acid/base. Mammalian TRs require selenocysteine (Sec) incorporated into the Gly-Cys-Sec-Gly-OH (GCUG) C-terminal tetrapeptide motif, while the TR from Drosophila melanogaster (DmTR) does not, and instead contains a Ser-Cys-Cys-Ser-OH (SCCS) tetrapeptide motif indicating that Sec is not universally necessary to catalyze the reduction of thioredoxin. This project has achieved three major objectives; 1) development of a semisynthetic method for production of mouse mitochondrial TR (mTR3) for structure-function studies, 2) establishment of a new method to study the mechanism of TR by using tetrapeptides in the oxidized form equivalent to the C-terminal active sites as substrates for the truncated forms of both enzymes, 3) determination of the crystal structure of DmTR. The results show that the structure of DmTR explains the biochemical data and has developed a new testable hypothesis in the field for the requirement of Sec in mammalian TR. We demonstrate that the tetrapeptides tested in Aim 2 were all better substrates for DmTR. The data also shows a far greater dependence on Sec for mTR3 than DmTR, which is in agreement with that observed for the collection full-length mutants produced for each enzyme in Aim 1. As this method of investigation is more analogous to the other enzymes of the GR family, the structures of the tetrapeptides determined by NMR spectroscopy were oriented in the active site of the both enzymes using the diglutathione bound in the structure of GR as template. DmTR appears to have a more open active site than observed in the known structure of mTR3. Residues from the helical face of the FAD-domain proximal to the FAD-associated active site are less bulky in DmTR to accommodate the hydroxyls of the serines. This is likely to make the enzyme more amenable for the conformational switching of the SCCS peptide necessary to protonate the leaving group cysteine by the proposed catalytic acid/base. In contrast, mTR3 shows a more restricted interface by incorporating bulkier residues at the interface in conjunction with the smaller Gly residues of the C-terminal sequence GCUG. The tetrapeptides display a conformational preference not suitable for protonation of the first leaving group in mTR3