Publications

• Association of ATP: glutamine synthetase adenylyltransferase activity with the P1 component of the glutamine synthetase deadenylylation system.

  Anderson W.B., Hennig S.B., Ginsburg A., Stadtman E.R.

  Regulation of glutamine synthetase (EC 6.3.1.2) in Escherichia coli is mediated by adenylylation and deadenylylation of the enzyme. The present studies show that one protein is a common component of both the adenylylation and deadenylylation systems. Thus, the ATP:glutamine synthetase adenylyltran ... >> More

  Regulation of glutamine synthetase (EC 6.3.1.2) in Escherichia coli is mediated by adenylylation and deadenylylation of the enzyme. The present studies show that one protein is a common component of both the adenylylation and deadenylylation systems. Thus, the ATP:glutamine synthetase adenylyltransferase, which catalyzes adenylylation of glutamine synthetase, and one of the two proteins required for deadenylylation (the P(I) protein) are inseparable by a variety of fractionation procedures. The adenylyltransferase and P(I)-deadenylylating activities behave as a single protein upon filtration through Agarose A 0.5 gel, and during chromatography on DE32 cellulose and hydroxyapatite columns. They migrate as a single protein band during electrophoresis on polyacrylamide gel and have identical susceptibilities to heat inactivation. These data indicate that the adenylyltransferase and the P(I)-deadenylylation activity are associated with the same protein complex. << Less

  Proc Natl Acad Sci U S A 67:1417-1424(1970) [PubMed] [EuropePMC]

• Expression, purification, crystallization, and preliminary X-ray analysis of the N-terminal domain of Escherichia coli adenylyl transferase.

  Xu Y., Wen D., Clancy P., Carr P.D., Ollis D.L., Vasudevan S.G.

  A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT ... >> More

  A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domain is truncated at the end of a predicted helix and prior to a Q-linker. The domain was found to be very soluble and stable so that it could be purified to homogeneity and crystallized. This construct has deadenylylation activity that is independent of the low nitrogen status indicator PII-UMP. The crystals belong to space group P3(1)21 or its enantiomorph P3(2)21 with a=b=116.6 A and c=67.6 A. << Less

  Protein Expr. Purif. 34:142-146(2004) [PubMed] [EuropePMC]

• The glutamine synthetase deadenylylating enzyme system from Escherichia coli. Resolution into two components, specific nucleotide stimulation, and cofactor requirements.

  Shapiro B.M.

  Biochemistry 8:659-670(1969) [PubMed] [EuropePMC]

• Reversible adenylylation of glutamine synthetase is dynamically counterbalanced during steady-state growth of Escherichia coli.

  Okano H., Hwa T., Lenz P., Yan D.

  Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by sma ... >> More

  Glutamine synthetase (GS) is the central enzyme for nitrogen assimilation in Escherichia coli and is subject to reversible adenylylation (inactivation) by a bifunctional GS adenylyltransferase/adenylyl-removing enzyme (ATase). In vitro, both of the opposing activities of ATase are regulated by small effectors, most notably glutamine and 2-oxoglutarate. In vivo, adenylyltransferase (AT) activity is critical for growth adaptation when cells are shifted from nitrogen-limiting to nitrogen-excess conditions and a rapid decrease of GS activity by adenylylation is needed. Here, we show that the adenylyl-removing (AR) activity of ATase is required to counterbalance its AT activity during steady-state growth under both nitrogen-excess and nitrogen-limiting conditions. This conclusion was established by studying AR(-)/AT(+) mutants, which surprisingly displayed steady-state growth defects in nitrogen-excess conditions due to excessive GS adenylylation. Moreover, GS was abnormally adenylylated in the AR(-) mutants even under nitrogen-limiting conditions, whereas there was little GS adenylylation in wild-type strains. Despite the importance of AR activity, we establish that AT activity is significantly regulated in vivo, mainly by the cellular glutamine concentration. There is good general agreement between quantitative estimates of AT regulation in vivo and results derived from previous in vitro studies except at very low AT activities. We propose additional mechanisms for the low AT activities in vivo. The results suggest that dynamic counterbalance by reversible covalent modification may be a general strategy for controlling the activity of enzymes such as GS, whose physiological output allows adaptation to environmental fluctuations. << Less

  J Mol Biol 404:522-536(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and functional roles of the P I and P II components of Escherichia coli glutamine synthetase deadenylylation system.

  Anderson W.B., Stadtman E.R.

  Arch. Biochem. Biophys. 143:428-443(1971) [PubMed] [EuropePMC]

• Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase.

  Xu Y., Zhang R., Joachimiak A., Carr P.D., Huber T., Vasudevan S.G., Ollis D.L.

  We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. ... >> More

  We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding. << Less

  Structure 12:861-869(2004) [PubMed] [EuropePMC]

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

  Jaggi R., van Heeswijk W.C., Westerhoff H.V., Ollis D.L., Vasudevan S.G.

  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 deadenylylatio ... >> More

  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. << Less

  EMBO J. 16:5562-5571(1997) [PubMed] [EuropePMC]

• Glutamine synthetase deadenylation: a phosphorolytic reaction yielding ADP as nucleotide product.

  Anderson W.B., Stadtman E.R.

  Biochem. Biophys. Res. Commun. 41:704-709(1970) [PubMed] [EuropePMC]