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. 1997 Sep 15;16(18):5562–5571. doi: 10.1093/emboj/16.18.5562

The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction.

R Jaggi 1, W C van Heeswijk 1, H V Westerhoff 1, D L Ollis 1, S G Vasudevan 1
PMCID: PMC1170188  PMID: 9312015

Abstract

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.

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Selected References

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  1. Adler S. P., Purich D., Stadtman E. R. Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme. J Biol Chem. 1975 Aug 25;250(16):6264–6272. [PubMed] [Google Scholar]
  2. Anderson W. B., Stadtman E. R. Glutamine synthetase deadenylation: a phosphorolytic reaction yielding ADP as nucleotide product. Biochem Biophys Res Commun. 1970 Nov 9;41(3):704–709. doi: 10.1016/0006-291x(70)90070-7. [DOI] [PubMed] [Google Scholar]
  3. Bueno R., Pahel G., Magasanik B. Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli. J Bacteriol. 1985 Nov;164(2):816–822. doi: 10.1128/jb.164.2.816-822.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caban C. E., Ginsburg A. Glutamine synthetase adenylyltransferase from Escherichia coli: purification and physical and chemical properties. Biochemistry. 1976 Apr 6;15(7):1569–1580. doi: 10.1021/bi00652a030. [DOI] [PubMed] [Google Scholar]
  5. Carr P. D., Cheah E., Suffolk P. M., Vasudevan S. G., Dixon N. E., Ollis D. L. X-ray structure of the signal transduction protein from Escherichia coli at 1.9 A. Acta Crystallogr D Biol Crystallogr. 1996 Jan 1;52(Pt 1):93–104. doi: 10.1107/S0907444995007293. [DOI] [PubMed] [Google Scholar]
  6. Cárdenas M. L., Cornish-Bowden A. Characteristics necessary for an interconvertible enzyme cascade to generate a highly sensitive response to an effector. Biochem J. 1989 Jan 15;257(2):339–345. doi: 10.1042/bj2570339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elvin C. M., Thompson P. R., Argall M. E., Hendry P., Stamford N. P., Lilley P. E., Dixon N. E. Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene. 1990 Mar 1;87(1):123–126. doi: 10.1016/0378-1119(90)90503-j. [DOI] [PubMed] [Google Scholar]
  8. Goldbeter A., Koshland D. E., Jr Ultrasensitivity in biochemical systems controlled by covalent modification. Interplay between zero-order and multistep effects. J Biol Chem. 1984 Dec 10;259(23):14441–14447. [PubMed] [Google Scholar]
  9. Hennig S. B., Ginsburg A. ATP: glutamine synthetase adenylytransferase from Escherichia coli: purification and properties of a low-molecular weight enzyme form. Arch Biochem Biophys. 1971 Jun;144(2):611–627. doi: 10.1016/0003-9861(71)90368-7. [DOI] [PubMed] [Google Scholar]
  10. Holm L., Sander C. DNA polymerase beta belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem Sci. 1995 Sep;20(9):345–347. doi: 10.1016/s0968-0004(00)89071-4. [DOI] [PubMed] [Google Scholar]
  11. Jaggi R., Ybarlucea W., Cheah E., Carr P. D., Edwards K. J., Ollis D. L., Vasudevan S. G. The role of the T-loop of the signal transducing protein PII from Escherichia coli. FEBS Lett. 1996 Aug 5;391(1-2):223–228. doi: 10.1016/0014-5793(96)00737-5. [DOI] [PubMed] [Google Scholar]
  12. Kahn D., Westerhoff H. V. Control theory of regulatory cascades. J Theor Biol. 1991 Nov 21;153(2):255–285. doi: 10.1016/s0022-5193(05)80426-6. [DOI] [PubMed] [Google Scholar]
  13. Kamberov E. S., Atkinson M. R., Ninfa A. J. The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J Biol Chem. 1995 Jul 28;270(30):17797–17807. doi: 10.1074/jbc.270.30.17797. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Love C. A., Lilley P. E., Dixon N. E. Stable high-copy-number bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene. 1996 Oct 17;176(1-2):49–53. doi: 10.1016/0378-1119(96)00208-9. [DOI] [PubMed] [Google Scholar]
  16. Magasanik B. The regulation of nitrogen utilization in enteric bacteria. J Cell Biochem. 1993 Jan;51(1):34–40. doi: 10.1002/jcb.240510108. [DOI] [PubMed] [Google Scholar]
  17. Muse W. B., Bender R. A. Map position of the glnE gene from Escherichia coli. J Bacteriol. 1992 Dec;174(23):7876–7877. doi: 10.1128/jb.174.23.7876-7877.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rhee S. G., Park R., Chock P. B., Stadtman E. R. Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3138–3142. doi: 10.1073/pnas.75.7.3138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Son H. S., Rhee S. G. Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene. J Biol Chem. 1987 Jun 25;262(18):8690–8695. [PubMed] [Google Scholar]
  21. Vasudevan S. G., Gedye C., Dixon N. E., Cheah E., Carr P. D., Suffolk P. M., Jeffrey P. D., Ollis D. L. Escherichia coli PII protein: purification, crystallization and oligomeric structure. FEBS Lett. 1994 Jan 17;337(3):255–258. doi: 10.1016/0014-5793(94)80203-3. [DOI] [PubMed] [Google Scholar]
  22. Westerhoff H. V., van Heeswijk W., Kahn D., Kell D. B. Quantitative approaches to the analysis of the control and regulation of microbial metabolism. Antonie Van Leeuwenhoek. 1991 Oct-Nov;60(3-4):193–207. doi: 10.1007/BF00430365. [DOI] [PubMed] [Google Scholar]
  23. Wootton J. C., Drummond M. H. The Q-linker: a class of interdomain sequences found in bacterial multidomain regulatory proteins. Protein Eng. 1989 May;2(7):535–543. doi: 10.1093/protein/2.7.535. [DOI] [PubMed] [Google Scholar]

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