No abstract

Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with ␥-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

An enzymatic method for synthesizing various ␥-D-glutamyl compounds efficiently and stereospecifically involving bacterial ␥-glutamyltranspeptidase (EC 2.3.2.2) with D-glutamine as a ␥-glutamyl donor was developed. With D-glutamine as a ␥-glutamyl donor instead of L-glutamine in ␥-glutamyltaurine synthesis, byproducts such as ␥-glutamylglutamine and ␥-glutamyl-␥-glutamyltaurine were not synthesized and the yield of ␥-glutamyltaurine dramatically increased from 25 to 71%. It was also shown that the purification could be simplified without these ␥-glutamyl by-products. The possibility of synthesizing various ␥-D-glutamyl compounds was also shown.␥-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) catalyzes the hydrolysis of ␥-glutamyl compounds such as glutathione and the transfer of their ␥-glutamyl moieties to other amino acids and peptides (27). The reactions catalyzed by GGT proceed via a ␥-glutamyl enzyme intermediate involving the hydroxyl group of . If the intermediate is subjected to nucleophilic attack by water, the reaction is hydrolytic, releasing glutamic acid. If the intermediate is subjected to nucleophilic attack by amino acids and peptides, the reaction is a transpeptidation yielding new ␥-glutamyl compounds. By employing various ␥-glutamyl acceptors, we can synthesize various ␥-glutamyl compounds using GGT. The pH optima for these reactions are very different (25). Therefore, by adjusting the pH of the reaction mixture, we can make GGT catalyze the transpeptidation reaction selectively. ␥-Glutamyl compounds are very attractive, because (i) compounds that are not very soluble in water become much more soluble with ␥-glutamylization (5); (ii) the ␥-glutamyl linkage is resistant to peptidases in serum, and some ␥-glutamyl compounds can possibly be used as prodrugs specific for the organs that express GGT (8,10,18,30); and (iii) some ␥-glutamyl compounds taste good and thus can be used as food additives (21,22). We have already developed and reported efficient methods for synthesizing various ␥-Lglutamyl compounds using Escherichia coli 21,22,26). The characteristics of our methods are as follows. (i) L-Glutamine, which is less expensive than glutathione, can be used as a ␥-glutamyl donor. (ii) No energy source, such as ATP, is required because GGT is a transferase and not a synthetase. (iii) Since E. coli GGT exhibits a broad substrate specificity for ␥-glutamyl acceptors, various ␥-glutamyl compounds can be synthesized. (iv) Since E. coli GGT can be purified from overproducing strains by means of a simple twostep method (1, 23), a large amount of GGT is readily available.During the development of a method for the enzymatic production of various ␥-glutamyl compounds, we found that the yield of a ␥-glutamyl compound with a ␥-glutamyl acceptor such as taurine was not very high. This was mainly because nonnegligible amounts of by-products, such as ␥-glutamylglutamine and ␥-glutamyl-␥-glutamyltaurine, were formed (26). It has been reported that GGT cannot utilize D-amino acids as ␥-glutamyl acceptors (27-29). Th...