Structural and mechanistic insights into unusual thiol disulfide oxidoreductase

Abstract

Cytoplasmic desulfothioredoxin (Dtrx) from the anaerobe Desulfovibrio vulgaris Hildenborough has been identified as a new member of the thiol disulfide oxidoreductase family. The active site of Dtrx contains a particular consensus sequence, CPHC, never seen in the cytoplasmic thioredoxins and generally found in periplasmic oxidases. Unlike canonical thioredoxins (Trx), Dtrx does not present any disulfide reductase activity, but it presents instead an unusual disulfide isomerase activity. We have used NMR spectroscopy to gain insights into the structure and the catalytic mechanism of this unusual Dtrx. The redox potential of Dtrx (-181 mV) is significantly less reducing than that of canonical Trx. A pH dependence study allowed the determination of the pK(a) of all protonable residues, including the cysteine and histidine residues. Thus, the pK(a) values for the thiol group of Cys(31) and Cys(34) are 4.8 and 11.3, respectively. The His(33) pK(a) value, experimentally determined for the first time, differs notably as a function of the redox states, 7.2 for the reduced state and 4.6 for the oxidized state. These data suggest an important role for His(33) in the molecular mechanism of Dtrx catalysis that is confirmed by the properties of mutant DtrxH33G protein. The NMR structure of Dtrx shows a different charge repartition compared with canonical Trx. The results presented are likely indicative of the involvement of this protein in the catalysis of substrates specific of the anaerobe cytoplasm of DvH. The study of Dtrx is an important step toward revealing the molecular details of the thiol-disulfide oxidoreductase catalytic mechanism.

FIGURE 1.

Dtrx activities. A, insulin reduction assay by Trx1 and Dtrx as a function of pH. The disulfide reductase activity was determined by using the insulin reduction assay. The assay was performed at 306 K with 2 μm Dtrx (triangle) or 2 μm Trx1 (black circle). The catalyzed reduction of insulin (100 μm) was followed by measuring an increase in absorbance at 650 nm and evaluated at different pH levels: 4.22, 6.3, 7.06, 7.52, 8.11, and 8.98. The absorbance caused by the nonenzymatic insulin reduction by DTT (1 mm) is substracted. B, scrambled RNase A (ScRNase) refolding assay: yield of RNase A activity of native RNase A and reshuffling of ScRNase A after incubation with DsbC, Trx1, Dtrx, and Dtrx H33G mutant. For the determination of a percentage of the RNase A activity, the mean intensity of several isolated peaks in RNA spectrum was used relative to the RNA spectrum in the presence of native RNase A. The RNA spectrum in presence of ScRNase A is used as blank. The error analysis of the data points was performed using Excel software. C, study of in vivo activity. Qualitative visualization of PalB activity using tributyrin plate for recombinant E. coli TG1 and Rosetta-gami as host cells. Different expression systems were observed: pJF119EH PalB (a), pJF119EH Dtrx-PalB (b), pJF119EH Trx1-PalB (c) in E. coli TG1 and pJF119EH PalB (d) and pJF119EH Dtrx-PalB (e) in E. coli Rosetta-gami.

FIGURE 2.

Titration of the redox potential of the disulfide bond of Dtrx and DtrxH33G. Changes in NMR signal intensities for the NH resonances of Cys³⁴ under oxidizing or reducing conditions were reported in the function of half-cell potential of glutathione. Gray triangles and black circles represent signal intensity in the oxidized and reduced states, respectively. Redox potentials were calculated using the Nernst equation from the ratio of concentrations of reduced and oxidized glutathione. Experimental data were fitted against a sigmoidal decay (logistic) function.

FIGURE 3.

pK_a determination of all Dtrx ionizable residues. A, 600 MHz two-dimensional CBCACO spectrum of reduced Dtrx at 298 K, pH 5.7, showing the cross-peaks for the C_α-CO and C_β-CO of all residues of the protein. Cross-peaks for the C_α-C_γ and C_β-C_γ of Asn and Asp, and for the C_β-C_δ and C_γ-C_δ of Gln and Glu are also visible and connected by lines. The inset shows a close-up view of the pH-dependent chemical shift variations for the C_β-CO of Asp²¹ (pH 4.9 (pink), 5.6 (red), 6.1 (orange), 6.6 (yellow), 7 (green), 7.8 (blue), and 9.3 (purple)). B, pK_a determination of the nucleophilic cysteine Cys³¹ (black circle) and cysteine Cys³⁴ (white circle) in the reduced form of Dtrx. C, pK_a determination of the histidine His³³ in the oxidized form (white triangle) and reduced form (black circle) of Dtrx. The pH-dependent chemical shift variation of the C_β carbons was measured, normalized, and fitted to one apparent pK_a value using the Henderson-Hasselbach equation.

FIGURE 4.

Three-dimensional structure of Dtrx in both redox states. A, overlay of the three-dimensional solution structures of the reduced (green) and oxidized (red) forms of Dtrx calculated with CYANA (21). The NMR sample contained 1 mm protein concentration (90% H₂O, 10% D₂O) in 100 mm NaCl, 50 mm phosphate buffer, pH 5.7, at 290 K. For reduced Dtrx, the intramolecular disulfide bond was reduced by adding DTT to a final concentration of 10 mm, under argon atmosphere. The Dtrx typical thioredoxin fold is represented in cartoon, and the side chains of the cysteine residues are shown in sticks. B, local conformations of the active site in the reduced form of Dtrx. C, local conformations of the active site in the oxidized form of Dtrx. The active site residues Cys³¹, Pro³², His³³, and Cys³⁴ are shown and labeled. Sulfur atoms are shown in yellow, hydrogen atoms are in white, and nitrogen and oxygen atoms are in blue and red, respectively. The figures were generated using the PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.

FIGURE 5.

Comparative structural analysis of Dtrx. A, electrostatic surface potential representations of reduced Dtrx and reduced E. coli Trx, with blue representing basic residues, red representing acidic residues, and white representing neutral residues. The orientations of Dtrx and Trx are similar. The position of the sulfur atom of the N-terminal cysteine is indicated by a star. The hydrophobic patch observed in Trx is not conserved in Dtrx. Surface calculations were made using MolMol. B, superimposition of the redox active site of the reduced form of Dtrx (green) and E. coli DsbA (pink) in stick representation. The active site residues are labeled. C, sequence alignment of Dtrx, E. coli Trx, E. coli DsbA, and E. coli DsbC. Identical residues are in red boxes. Conserved residues are shown in red. The alignment was prepared with TCoffee (34) and ESPript (11).

FIGURE 6.

Model of the Dtrx catalytic mechanism. Substrate reduction (solid lines) by Dtrx occurs in four steps. In the first step, the Cys³¹ thiolate of Dtrx nucleophilically attacks sulfur atom of the substrate disulfide. In the second step, the thiolate of the substrate produces a base attack on Cys³⁴ sulfur atom. Next, the Cys³⁴ thiolate attacks the Cys³¹ sulfur atom involved in the disulfide bond, causing the rupture of the latter. At the same time, the imidazole group of His³³ is deprotonated by a base attack of the thiolate of the reduced substrate. This reaction produces an oxidized Dtrx and a reduced dithiol substrate. Substrate oxidation (dotted lines) by Dtrx occurs in four steps. First, the deprotonated His³³ produces a base attack on the first cysteine of the substrate. After, this activated cysteine of the substrate nucleophilically attacks the sulfur atom of Cys³¹ of the oxidized Dtrx, leading to the formation of a disulfide-linked complex between Dtrx and the substrate. In the next step, the second cysteine of the substrate is deprotonated probably by the Cys³⁴ thiolate of Dtrx and attacks the sulfur atom of the substrate cysteine, which is disulfide-bonded with Cys³¹ of Dtrx. This reaction results in the formation of a disulfide bond in the substrate and the reduction of Dtrx with a protonated His³³.