Thioredoxin system from Deinococcus radiodurans

Abstract

This paper describes the cloning, purification, and characterization of thioredoxin (Trx) and thioredoxin reductase (TrxR) and the structure determination of TrxR from the ionizing radiation-tolerant bacterium Deinococcus radiodurans strain R1. The genes from D. radiodurans encoding Trx and TrxR were amplified by PCR, inserted into a pET expression vector, and overexpressed in Escherichia coli. The overexpressed proteins were purified by metal affinity chromatography, and their activity was demonstrated using well-established assays of insulin precipitation (for Trx), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reduction, and insulin reduction (for TrxR). In addition, the crystal structure of oxidized TrxR was determined at 1.9-A resolution. The overall structure was found to be very similar to that of E. coli TrxR and homodimeric with both NADPH- and flavin adenine dinucleotide (FAD)-binding domains containing variants of the canonical nucleotide binding fold, the Rossmann fold. The K(m) (5.7 muM) of D. radiodurans TrxR for D. radiodurans Trx was determined and is about twofold higher than that of the E. coli thioredoxin system. However, D. radiodurans TrxR has a much lower affinity for E. coli Trx (K(m), 44.4 muM). Subtle differences in the surface charge and shape of the Trx binding site on TrxR may account for the differences in recognition. Because it has been suggested that TrxR from D. radiodurans may have dual cofactor specificity (can utilize both NADH and NADPH), D. radiodurans TrxR was tested for its ability to utilize NADH as well. Our results show that D. radiodurans TrxR can utilize only NADPH for activity.

FIG. 1.

SDS-PAGE of purified samples of various TrxR and Trx proteins, as follows: (1) E. coli thioredoxin (Trx), (2) D. radiodurans thioredoxin (Trx1), (3) low-molecular-mass protein marker, (4) E. coli thioredoxin reductase (TrxR), and (5) D. radiodurans thioredoxin reductase (TrxR).

FIG. 2.

Sequence alignment of thioredoxins from E. coli and D. radiodurans. The N terminus of D. radiodurans Trx1 is more similar to that of E. coli Trx2 than that of E. coli Trx1. Absolutely conserved active-site cysteines are indicated with green asterisks, whereas N terminus-conserved cysteines of D. radiodurans Trx1 and E. coli Trx2 are indicated with blue asterisks. The figure was produced using the program ClustalW version 2.0 (18) and the ESPript server (12).

FIG. 3.

Michaelis-Menten plot of D. radiodurans TrxR, determined using D. radiodurans Trx1 as a substrate. The reaction was monitored by consumption of NADPH via a decrease in absorbance at 340 nm. Initial velocity (V_o) was measured as μmoles of NADPH consumed per minute. Reaction mixtures contained 100 mM HEPES buffer (pH 7.4), 2 mM EDTA, 30 μM solution of bovine insulin (Sigma), 0.1 mM NADPH (Calbiochem), 0.1 μM D. radiodurans TrxR, and D. radiodurans Trx1 (0 to 35 μM).

FIG. 4.

Sequence alignment of TrxR proteins from E. coli and D. radiodurans. The conserved active-site cysteine residues are indicated with green asterisks, the key residues involved in interaction with Trx are indicated with blue asterisks, and the GXGXXA motifs are indicated with red asterisks. The figure was produced using the program ClustalW version 2.0 (18) and the ESPript server (12).

FIG. 5.

Overall structure of the A chain of D. radiodurans TrxR in the FO conformation. In this conformation, the FAD-binding domain is too far from the NADPH-binding domain for electron transfer to occur from NADPH to FAD. A 67° rotation by the NADPH-binding domain brings NADPH close to FAD, allowing electron transfer to occur from NADPH to FAD (21). The figure was generated by PYMOL (9).

FIG. 6.

Superimposed TrxR structures from E. coli (red) and D. radiodurans (blue), showing some key residues that are important for binding to thioredoxin. The figure was generated by PYMOL (9).

FIG. 7.

Electrostatic potentials of TrxR and Trx from E. coli (A and B, respectively) and D. radiodurans (C and D, respectively), calculated by APBS software (4). Positively and negatively charged areas are colored in red and in blue and contoured at −5 k_BTe⁻¹ and +5 k_BTe⁻¹, respectively. Key residues of the TrxR-Trx complex interface are shown on the diagram. The overall shapes of the Trx-binding sites differ between the two TrxR proteins, but there are only small differences in the electrostatic potentials. The figures were generated with PYMOL (9) using E. coli TrxR, E. coli Trx, and D. radiodurans TrxR coordinates (PDB accession no. 1TDE, 2TRX, and 2Q7V, respectively). The D. radiodurans Trx figure was generated using the D. radiodurans Trx homology model.