Novel Structure, Properties and Inactivation of Glutamine Synthetase Cloned from Bacteroides fragilis

• Published in: Journal of general microbiology Vol. 133; no. 9; pp. 2437 - 2446
• Main Authors: SOUTHERN, JAMES A, PARKER, JOAN R, WOODS, DAVID R
• Format: Journal Article
• Language: English
• Published: London Soc General Microbiol 01.09.1987; New York, NY Cambridge University Press
• Subjects: Bacteriology; Bacteroides fragilis; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; glutamate-ammonia ligase; Metabolism. Enzymes; Microbiology; Regulation(control); Gene; Molecular structure; Bacteroides fragilis; Enzyme; Escherichia coli; Bacteria; Glutamine synthetase; Molecular cloning; Escherichieae; Bacteroidaceae; Enterobacteriaceae
• ISSN: 0022-1287; 1350-0872; 1465-2080
• DOI: 10.1099/00221287-133-9-2437

1 State Vaccine Institute, Alexandra Road, Pinelands 7405, South Africa 2 Department of Microbiology, University of Cape Town, Rondebosch 7700, South Africa ABSTRACT SUMMARY: The cloned Bacteroides fragilis glutamine synthetase (GS) subunit produced in Escherichia coli had the same apparent M r of approximately 75000 as the GS subunit from B. fragilis cells. The B. fragilis GS enzyme had an apparent M r of approximately 490000 and it is concluded that the GS is a hexamer. The cloned GS did not appear to be regulated by adenylylation and deadenylylation and the cloned enzyme was inactivated by snake venom phosphodiesterase. The pH profiles of the cloned GS, assayed by the -glutamyl transferase (GGT) assay were similar for NH 4 + -shocked and unshocked cell extracts and an isoactivity point was not obtained from these curves. The cloned GS was subject to feedback inhibition by amino acids but not by AMP. The GGT activity of the cloned GS in NH 4 + -shocked and unshocked cell-free extracts was inhibited by Mg 2+ . Mn 2+ stimulated the cloned GS GGT activity of NH 4 + -shocked cell-free extracts. Western blotting indicated that GS production was regulated by nitrogen in B. fragilis cells but cell extracts showed no GGT activity. Cloned B. fragilis GS produced in E. coli was specifically and irreversibly inactivated by B. fragilis cell extracts.