␥-Glutamyltranspeptidase (GGT) is a heterodimic enzyme that is generated from the precursor protein through posttranslational processing and catalyzes the hydrolysis of ␥-glutamyl bonds in ␥-glutamyl compounds such as glutathione and͞or the transfer of the ␥-glutamyl group to other amino acids and peptides. We have determined the crystal structure of GGT from Escherichia coli K-12 at 1.95 Å resolution. GGT has a stacked ␣␤␤␣ fold comprising the large and small subunits, similar to the folds seen in members of the N-terminal nucleophile hydrolase superfamily. The active site Thr-391, the N-terminal residue of the small subunit, is located in the groove, from which the pocket for ␥-glutamyl moiety binding follows. We have further determined the structure of the ␥-glutamyl

Prevention of quality of life (QOL) deterioration is associated with the inhibition of geriatric diseases and the regulation of brain function. However, no substance is known that prevents the aging of both body and brain. It is known that polyamine concentrations in somatic tissues (including the brain) decrease with increasing age, and polyamine-rich foods enhance longevity in yeast, worms, flies, and mice, and protect flies from age-induced memory impairment. A main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal bacteria. We found that arginine intake increased the concentration of putrescine in the colon and increased levels of spermidine and spermine in the blood. Mice orally administered with arginine in combination with the probiotic bifidobacteria LKM512 long-term showed suppressed inflammation, improved longevity, and protection from age-induced memory impairment. This study shows that intake of arginine and LKM512 may prevent aging-dependent declines in QOL via the upregulation of polyamines.

y-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity. The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B). The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000. The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000. Fraction A catalyzes the hydrolysis and transpeptidation of all -y-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors. The apparent Km values for glutathione and y-glutamyl-p-nitroanilide as y-glutamyl donors in the transpeptidation reaction were both 35 ,uM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively. The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT. The temperature stability of the purified GGT supports our hypothesis that E. coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.

A novel bacterial putrescine utilization pathway was discovered. Seven genes, the functions of whose products were not known, are involved in this novel pathway. Five of them encode enzymes that catabolize putrescine; one encodes a putrescine importer, and the other encodes a transcriptional regulator. This novel pathway involves six sequential steps as follows: 1) import of putrescine; 2) ATP-dependent ␥-glutamylation of putrescine; 3) oxidization of ␥-glutamylputrescine; 4) dehydrogenation of ␥-glutamyl-␥-aminobutyraldehyde; 5) hydrolysis of the ␥-glutamyl linkage of ␥-glutamyl-␥-aminobutyrate; and 6) transamination of ␥-aminobutyrate to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate.

␥-Glutamyltranspeptidase is the key enzyme in glutathione metabolism, and we previously presented evidence suggesting that it belongs to the N-terminal nucleophile hydrolase superfamily. Enzymatically active ␥-glutamyltranspeptidase, which consists of one large subunit and one small subunit, is generated from an inactive common precursor through post-translational proteolytic processing. The processing mechanism for ␥-glutamyltranspeptidase of Escherichia coli K-12 has been analyzed by means of in vitro studies using purified precursors. Here we show that the processing of a precursor of ␥-glutamyltranspeptidase is an intramolecular autocatalytic event and that the catalytic nucleophile for the processing reaction is the oxygen atom of the side chain of Thr-391 (N-terminal residue of the small (␤) subunit), which is also the nucleophile for the enzymatic reaction.

Glutamate-putrescine ligase (␥-glutamylputrescine synthetase, PuuA, EC 6.3.1.11) catalyzes the ␥-glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving ␥-glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent ␥-glutamylation of putrescine. The K m values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mM, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without ␥-glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway.

Some amino acids and peptides, which have low solubility in water, become much more soluble following gamma-glutamylation. Compounds become more stable in the blood stream with gamma-glutamylation. Several gamma-glutamyl compounds are known to have favorable physiological effects on mammals. Gamma-glutamylation can improve taste and can stabilize glutamine in aqueous solution. Because of such favorable features, gamma-glutamyl compounds are very attractive. However, only a small number of gamma-glutamyl amino acids have been studied although many other gamma-glutamyl compounds may have characteristics that will benefit humans. This is mainly because gamma-glutamyl compounds have not been readily available. An efficient and simple method of producing various gamma-glutamyl compounds, especially gamma-glutamyl amino acids, using bacterial gamma-glutamyltranspeptidase has been developed. With this method, modifications of reactive groups of the substrate and energy source such as ATP are not required, and a wide-range of gamma-glutamyl compounds can be synthesized. Moreover, bacterial gamma-glutamyltranspeptidase, a catalyst for this method, is readily available from the strain over-producing this enzyme. The superiority of producing gamma-glutamyl compounds with bacterial gamma-glutamyltranspeptidase over other methods of production is discussed.

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