gamma-Glutamyltranspeptidase from Escherichia coli K-12: formation and localization

Abstract

Escherichia coli cells showed maximum activity of y-glutomyltranspeptidase (EC 2.3.2.2) when they were grown at 20°C, 14% of maximum activity at 37°C, and none at 43°C. The enzyme activity of intact cells grown at 20°C was stably maintained after the temperature was changed to 45°C. The activity increased during the exponential phase, and maximum activity was found at stationary phase. Its intracellular localization in the periplasmic space was confirmed.-y-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of -y-glutamyl compounds (hydrolyase activity glutaminase activity) and the transfer of their -y-glutamyl moieties to other amino acids and peptides (transferase activity) (24). Although a number of studies on GGT of mammalian cells have been performed and reviewed (1,32,33), the physiological roles of the enzyme are still controversial. GGT of yeasts was also investigated (15,26).Since Talalay (31) found GGT activity in Proteus vulgaris, several studies have been performed on GGT of bacteria, including an L-glutamate-fermenting bacterium (6) and Bacillus natto (2). Nakayama et al. in our laboratory reported that P. mirabilis exhibits high GGT activity (17), and then they purified and characterized it (18) and determined its localization in the cell (19). They also synthesized yglutamyl-L-3,4-dihydroxyphenylalanine (DOPA) enzymatically, using the GGT from P. mirabilis (16), and Togari et al. suggested that this y-glutamyl-L-DOPA is useful as a precursor of L-DOPA in rat brain (manuscript in preparation). Kiuchi et al. (9) also showed that this -y-glutamyl-L-DQPA is useful as a substrate for measuring the enzymatic activity of GGT.Milbauer and Grossowicz (12), Szewczuk and Mulczyk (29), and Nakayama et al. (20) found GGT activity in various bacteria, but they also found that Escherichia coli, the best-studied bacteria in laboratories, does not exhibit strong GGT activity.In this study we found that the GGT activity of E. coli K-12 is greatly affected by growth temperature and growth phase. The localization of this enzyme was also determined by cell fractionation involving osmotic shock or lysozyme treatment. MATERIALS AND METHODSReagents. y-Glutamyl-p-nitroanilide, glycylglycine, and bovine serum albumin were purchased from Wako Pure Chemical Co., lysozyme, o-nitrophenyl-,-D-galactopyranoside, and 5'-AMP were from Sigma Che,mical Co., tryptone was from Difco Laboratories, and yeast extract was from the Oriental Yeast Co. Other chemicals were the best reagents grade available from commercial sources.Bacterial strains and cultures. P. mirabilis was the same strain that Nakayama et al. described (18). Prototrophic E. coli K-12 MG1655 (4) was donated by Carol A. Gross. * Corresponding author. LB broth (13) was used unless otherwise stated. A temperature gradient incubator, model TN-3 (Toyo Kagaku Sangyo Co.), was used in an air-conditioned room at 28°C to create temperature gradients for cultures. In other cases, cells were grown in a 20°C water bath with reciprocal shaking. Precultures were grown at 37°C unle...

Increase of “Umami” and “Kokumi” Compounds in Miso, Fermented Soybeans, by the Addition of Bacterial γ-Glutamyltranspeptidase

“…GGT activity was measured by the standard assay method described previously (Suzuki, Kumagai, & Tochikura, 1986b). One unit of the enzyme was defined as the amount of enzyme that released 1 µmol of p-nitroaniline per minute from γ-glutamyl-p-nitroanilide (γ-GpNA).…”

Purification and Properties of Two Isozymes of γ-Glutamyltranspeptidase fromBacillus subtilisTAM-4

“…9 ) bacteria. Suzuki et al demonstrated that E. coli K-12 GGT was localized in the periplasmic space 10 ) and supposed that the major function of the enzyme was hydrolysis and not transpeptidation. 6 ) It is known that some strains of Bacillus l1 14) produce poly(y-glutamic acid).…”

Crystal structures of γ-glutamyltranspeptidase from Escherichia coli , a key enzyme in glutathione metabolism, and its reaction intermediate

“…There are, however, two notable differences between mammalian and E. coli enzymes. First, the precursor protein of E. coli GGT has a signal peptide at its N-terminal region (13) directing export to the periplasmic space (14), but mammalian GGTs have anchor domains in their N-terminal regions leading to membrane association (15). Additionally, mammalian GGTs are heterologously glycosylated, whereas E. coli GGT is nonglycosylated (2).…”