Crystallographic studies on redox enzymes containing the Thioredoxin fold

Abstract: Three-dimensional structures of two redox enzymes, glutathione peroxidase from human plasma and a protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus, have been determined by X-ray crystallography. Glutathione peroxidases constitute a family of antioxidant seleno-enzymes that catalyze the reduction of a variety of hydroperoxides. Selenocysteine is the active site residue of these enzymes, which presumably shuttles between the selenolate anion state and the selenenic acid state in the catalytic cycle. They usually use glutathione as the reducing substrate. Compared to the other members of this protein family, glutathione peroxidase from human plasma is the only identified extracellular enzyme, whose physiological function remains ambiguous due to the low levels of reduced glutathione in human plasma and the low reactivity of this enzyme. The plasma enzyme was crystallized and the structure was solved by Patterson search methods. Crystallographic refinement of the structure at 2.9 A resolution resulted in a R-factor of 0.222 and a free R-factor of 0.290. The structure of the plasma enzyme was compared with that of the bovine erythrocyte enzyme to understand their structural and functional differences. Like the bovine erythrocyte enzyme, the plasma enzyme shows the structural feature of a thioredoxin fold with a central ß sheet and flanking a helices. The active site selenocysteine is located in a loop preceding the first [alpha] helix. Distinct from the bovine erythrocyte enzyme, however, the plasma enzyme has an intrachain disulfide bridge and a few extra N-terminal residues which form an additional ß strand. Structural variations of residues in the active site region of the plasma enzyme may account for its low affinity for glutathione. Protein disulfide oxidoreductases belong to a superfamily of enzymes that catalyze the reduction, oxidation or rearrangement of protein disulfide bonds. The active site of these enzymes has a CXXC sequence motif, in which the two cysteines undergo reversible oxidation-reduction by shuttling between a dithiol and a disulfide in the catalytic process. The protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus is an enzyme that was first purified without a clearly defined physiological function although it showed the activity of catalyzing the reduction of protein disulfide bonds. The enzyme was crystallized and the structure was determined by multiple isomorphous replacement with anomalous scattering. The structure was refined to 1.9 A resolution with a R-factor of 0.192 and a free R-factor of 0.217. This is the first three-dimensional structure of an archaeal protein disulfide oxidoreductase. It reveals structural details that suggest a relation to eukaryotic protein disulfide isomerase. Compared to the known structures of other oxidoreductases, it shows the unique feature of having two adjacent homologous structural units with low sequence identity. Each unit contains a thioredoxin fold with an active site disulfide formed in a distinct CXXC motif. The two active site disulfides have different solvent accessibilities. They probably differ from each other in their conformational stabilities as well as their redox properties. The structural analysis forms the basis for further functional studies. It is very likely that the enzyme plays a broader functional role than a simple disulfide reductant as shown by recent biochemical experiments.