We report here the first three-dimensional structure of a mammalian thioltransferase as determined by single crystal X-ray crystallography at 2.2 A resolution. The protein is known for its thiol-redox properties and dehydroascorbate reductase activity. Recombinant pig liver thioltransferase expressed in Escherichia coli was crystallized in its oxidized form by vapor diffusion technique. The structure was determined by multiple isomorphous replacement method using four heavy-atom derivatives. The protein folds into an a / p structure with a four-stranded mixed &sheet in the core, flanked on either side by helices. The fold is similar to that found in other thiol-redox proteins, viz. E. coli thioredoxin and bacteriophage T4 glutaredoxin, and thus seems to be conserved in these functionally related proteins. The active site disulfide (Cys 22-Cys 25) is located on a protrusion on the molecular surface. Cys 22, which is known to have an abnormally low pK, of 3.8, is accessible from the exterior of the molecule. Pro 70, which is in close proximity to the disulfide bridge, assumes a conserved cis-peptide configuration. Mutational data available on the protein are in agreement with the three-dimensional structure.Keywords: crystal structure; dehydroascorbate reductase; disulfide; glutaredoxin; thiol oxidoreductase; thioltransferase Thiol-disulfide oxidoreductases, which include the thioltransferase, glutaredoxin, thioredoxin family of proteins, play a vital role in maintaining the redox status of sulfhydryl groups inside the cell and thus participate in catalyzing and/or regulating a variety of cellular functions (Holmgren et al., 1986;Wells et al., 1993). They typically transfer electrons from NADPH to the substrate in reactions coupled with other specific enzymes. Thioltransferase and glutaredoxin derive their reducing equivalents from glutathione, which in turn is linked to the glutathione reductase/NADPH system, whereas thioredoxin utilizes the thioredoxin reductase/NADPH pathway for the same purpose. Thioltransferase (Askelof et al., 1974) and glutaredoxin (Holmgren, 1976) were initially discovered due to different properties but were later found to be highly homologous in mammalian cells and in fact were shown to be one and the same protein by

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