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Dehydroascorbic acid is generated in plants and animal cells by oxidation of ascorbic acid. The reaction is believed to occur by the one-electron oxidation of ascorbic acid to semidehydroascorbate radical followed by disproportionation to dehydroascorbic acid and ascorbic acid. Semidehydroascorbic acid may recycle to ascorbic acid catalyzed by membrane-bound NADH-semidehydroscorbate reductase. However, disproportionation of the free radical occurs at a rapid rate, 10(5) M-1 s-1, accounting for measurable cellular levels of dehydroascorbate. Dehydroascorbate reductase, studied earlier and more extensively in plants, is now recognized as the intrinsic activity of thioltransferases (glutaredoxins) and protein disulfide isomerase in animal cells. These enzymes catalyze the glutathione-dependent two-electron regeneration of ascorbic acid. The importance of the latter route of ascorbic acid renewal was seen in studies of GSH-deficient rodents (Meister, A. (1992) Biochem. Pharmacol. 44, 1905-1915). GSH deficiency in newborn animals resulted in decreased tissue ascorbic acid and increased dehydroascorbate-to-ascorbate ratios. Administration of ascorbic acid daily to GSH-deficient animals decreased animal mortality and cell damage from oxygen stress. A cellular role is proposed for dehydroascorbate in the oxidation of nascent protein dithiols to disulfides catalyzed in the endoplasmic reticulum compartment by protein disulfide isomerase.

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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