Three-dimensional structure of a mammalian thioredoxin reductase: implications for mechanism and evolution of a selenocysteine-dependent enzyme

Abstract

Thioredoxin reductases (TrxRs) from mammalian cells contain an essential selenocysteine residue in the conserved C-terminal sequence Gly-Cys-SeCys-Gly forming a selenenylsulfide in the oxidized enzyme. Reduction by NADPH generates a selenolthiol, which is the active site in reduction of Trx. The three-dimensional structure of the SeCys498Cys mutant of rat TrxR in complex with NADP(+) has been determined to 3.0-A resolution by x-ray crystallography. The overall structure is similar to that of glutathione reductase (GR), including conserved amino acid residues binding the cofactors FAD and NADPH. Surprisingly, all residues directly interacting with the substrate glutathione disulfide in GR are conserved despite the failure of glutathione disulfide to act as a substrate for TrxR. The 16-residue C-terminal tail, which is unique to mammalian TrxR, folds in such a way that it can approach the active site disulfide of the other subunit in the dimer. A model of the complex of TrxR with Trx suggests that electron transfer from NADPH to the disulfide of the substrate is possible without large conformational changes. The C-terminal extension typical of mammalian TrxRs has two functions: (i) it extends the electron transport chain from the catalytic disulfide to the enzyme surface, where it can react with Trx, and (ii) it prevents the enzyme from acting as a GR by blocking the redox-active disulfide. Our results suggest that mammalian TrxR evolved from the GR scaffold rather than from its prokaryotic counterpart. This evolutionary switch renders cell growth dependent on selenium.

Figure 1

Stereo view of an F_o − F_c electron-density omit map for the C-terminal residues 484–499, contoured at 3.0 σ. The refined model is superimposed.

Figure 2

Amino acid sequence and secondary structure elements. The secondary structure of rat TrxR is shown in the upper line. The nomenclature of the secondary structure elements is the same as used for GR (31); each α-helix is labeled by a sequential number, and each β-strand is indicated by a letter giving the sheet name and a number. Second line, amino acid sequence of rat TrxR; third line, sequence of human GR. Conserved residues are shown in bold letters. Possible functions of individual residues are indicated in the lower line. f, flavin binding; n, NADPH binding; d, participation in dimer interface; a, active site.

Figure 3

Ribbon representation of the dimer of rat TrxR. The two subunits are shown in light or dark colors, respectively. Red, FAD binding domain; yellow, NADP binding domain; blue, interface domain. Bound FAD (red) and NADP (orange) are shown as ball-and-stick models.

Figure 4

Stereo view of the C-terminal extensions in enzymes of the pyridine nucleotide–disulfide oxidoreductase family. The structure of rat TrxR is shown as a ribbon model (color coding as in Fig. 3). The C-terminal extensions are shown as Cα traces (LiDH, dihydrolipoamide dehydrogenase, magenta; TrpR, trypanothione reductase, green; TrxR, rat TrxR, blue). The positions of the catalytic disulfide (Cys-59–Cys-64), and the cysteine residues Cys-497 and Cys-498 in the C-terminal tail of TrxR are indicated by yellow spheres.

Figure 5

Active site of the SeCys498Cys mutant of rat TrxR (color coding as in Fig. 3). The observed conformation of Tyr-200 shields FAD from the solvent and hinders proper binding of the nicotinamide ring of NADP⁺. The position of oxidized glutathione (Gl, brown) was derived from a superposition of the structures of rat TrxR and the complex of GR with glutathione (36). The positions of the sulfur atoms of glutathione, the catalytic disulfide (Cys-59–Cys-64), and the cysteine residues Cys-497 and Cys-498 are indicated by yellow spheres.

Figure 6

Model of the complex of rat TrxR with human Trx. Color coding for TrxR is as in Fig. 3, Trx is shown in green. Residues at the Trx–TrxR interface are shown as stick models. The positions of the sulfur atoms of the catalytic disulfide (Cys-59–Cys-64) and the cysteine residues Cys-32, Cys-35, Cys-497, and Cys-498 are indicated by yellow spheres.

Figure 7

Postulated reaction mechanism for mammalian TrxR.