Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade.

Enzymes and regulatory proteins involved in the cascade control of glutamine synthetase activity of Escherichia coli have been separated from one another and the effects of numerous metabolites on each step in the cascade have been determined. The adenylyl transferase (ATase) -catalyzed adenylylation of glutamine synthetase, which requires the presence of the unmodified form of the regulatory protein PII is enhanced by glutamine and is inhibited by either α-ketoglutarate (α-KG) or the uridylylated form (PII·UMP) of the regulatory protein. PII·UMP and α-KG act synergistically to inhibit this activity. In contrast, the PII·UMP-dependent, ATase-catalyzed deadenylylation of glutamine synthetase requires α-KG and ATP and is inhibited by glutamine or PII and synergistically by glutamine plus PII. The capacity of uridylyl transferase (UTase) to catalyze the uridylylation of PII is dependent on the presence of α-KG and ATP and is inhibited by glutamine. The deuridylylation of PII·UMP by the uridylyl removing enzyme (UR) is enhanced by glutamine but is unaffected by α-KG. However, CMP, UMP, and CoA all inhibit activity at 10^?6m. High concentrations of ATase inhibit both UR and UTase activities, presumably by binding the regulatory protein. Of more than 50 substances that alter the activity of at least one enzyme in the cascade, only α-KG and glutamine affect the activity at every step. This accounts for the observation that glutamine synthetase activity in vivo is very sensitive to the intracellular ratio of α-KG to glutamine.     

A source of ultrasensitivity in the glutamine response of the bicyclic cascade system controlling glutamine synthetase adenylylation state and activity in Escherichia coli

Glutamine synthetase (GS) activity in Escherichia coli is regulated by reversible adenylylation, brought about by a bicyclic system comprised of uridylyltransferase/uridylyl-removing enzyme (UTase/UR), its substrate, PII, adenylyltransferase (ATase), and its substrate, GS. The modified and unmodified forms of PII produced by the upstream UTase/UR-PII cycle regulate the downstream ATase-GS cycle. A reconstituted UTase/UR-PII-ATase-GS bicyclic system has been shown to produce a highly ultrasensitive response of GS adenylylation state to the glutamine concentration, but its composite UTase/UR-PII and ATase-GS cycles displayed moderate glutamine sensitivities when examined separately. Glutamine sensitivity of the bicyclic system was significantly reduced when the trimeric PII protein was replaced by a heterotrimeric form of PII that was functionally monomeric, and coupling between the two cycles was different in systems containing wild-type or heterotrimeric PII. Thus, the trimeric nature of PII played a role in the glutamine response of the bicyclic system. We therefore examined regulation of the individual AT (adenylylation) and AR (deadenylylation) activities of ATase by PII preparations with various levels of uridylylation. AR activity was affected in a linear fashion by PII uridylylation, but partially modified wild-type PII activated the AT much less than expected based on the extent of PII modification. Partially modified wild-type PII also bound to ATase less than expected based upon the fraction of modified subunits. Our results suggest that the AT activity is only bound and activated by completely unmodified PII and that this design is largely responsible for ultrasensitivity of the bicyclic system.     

In situ inactivation of E. coli uridylylation cycle is independent of the state of covalent modification of the components of the glutamine synthetase cascade

Permeabilization of nitrogen-starved cells of Escherichia coli W with Lubrol WX leads to a selective inactivation of the uridylyl-removing uridylyltransferase (UR/ UTase) enzyme of the glutamine synthetase (GS) cascade system; whereas similar treatment does not affect activity of UR/UTase in cells grown under conditions of nitrogen excess (10 mm glutamine) (Mura, U., and Stadtman, E. R. (1981) J. Biol. Chem.256, 13014–13021). The possibility that susceptibility to Lubrol inactivation is related to differences in the state of adenylylation of GS and/or in the state of uridylylation of the P_II protein was investigated. Permeabilized cells from nitrogen sufficient as well as from nitrogen-limited growth medium were exposed to Lubrol after prior incubation under conditions that lead to high or low states of GS adenylylation and high or low P_IID/P_IIA ratios. Integrity of UR/UTase was monitored by measuring the capacity of UTP to stimulate the deadenylylation of GS in situ. The results showed that the inactivation of UR/UTase by Lubrol is not affected by the states of GS adenylylation or P_II uridylylation.     

New methods for the colorimetric assay of PIID regulatory protein, uridylyltransferase, and uridylyl-removing enzyme in glutamine synthetase cascade

Adenylylation and deadenylylation of glutamine synthetase (GS) are catalyzed by the same adenylyltransferase (ATase). The ability of ATase to catalyze adenylylation is markedly stimulated by the unmodified form of a regulatory protein, P_IIA, whereas its capacity to catalyze deadenylylation is stimulated by the uridylylated form (P_IID) of the regulatory protein. Interconversion between P_IIA and P_IID is catalyzed by uridylyltransferase (UTase) and uridylylremoving enzyme (UR). New colorimetric methods were developed for the assays of P_IID, UTase, and UR activities. The P_IID activity is monitored by its unique ability to stimulate the ATase catalyzed formation of unadenylylated subunits from adenylylated GS. The inerease of unadenylylated subunits is determined by measuring the γ-glutamyltransferase activity of GS under conditions where the activity of an unadenylylated subunit is about 15 times greater than that of an adenylylated subunit (i.e., at pH 8.0 in the presence of Mn²⁺). Assays for UTase and UR enzyme are derived by coupling the P_IID assay to the UTase and UR reactions. For the UTase reaction, the formation of P_IID from P_IIA is measured, whereas the decrease in P_IID is followed for the UR assay. These assays have been applied to follow the activities of these proteins during their purification procedures, to the mechanistic studies on the deadenylylation reaction, and to determine the activities of these proteins in mutants produced during the genetic study of glutamine synthetase cascade. The problems evolved from these assays are discussed.     

Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme.

The PII regulatory protein of Escherichia coli glutamine synthetase exists in two interconvertible forms: a uridylylated form (PIID) which promotes the deadenylylation of glutamine synthetase and an unmodified form (PIIA) which promotes the adenylylation of glutamine synthetase (Mangum, J.H., Magni, G., and Stadtman, E.R. (1973) Arch. Biochem. Biophys. 158, 514-525). PII has been purified to homogeneity. Its molecular weight is 44,000. The protein is composed of four subunits, each with a molecular weight of approximately 11,000. The subunits are identical as judged by: (a) the homogeneity of the subunits in sodium dodecyl sulfate, 8 M urea, and 6 M guanidine HCl; (b) the minimal molecular weight calculated from the amino acid composition; and (c) the isolation of only two tryptic peptides containing tyrosine (there are 8 tyrosyl residues per 44,000 molecular species). Following iodination of PIIA and PIID with 125I in the presence of chloramine-T, tryptic digestion yields two radioactive peptides from PIIA and only one from PIID. Since a tyrosine with a substituted hydroxyl group cannot be iodinated, this result indicates that 1 tyrosyl residue in each subunit is modified by the covalent attachment of UMP. This conclusion is supported also by the fact that treatment of PIID with snake venom phosphodiesterase results in the release of covalently bound UMP and the stoichiometric appearance of phenolate ion (pH 13) as measured by ultraviolet absorption spectroscopy. The enzyme activities (uridylyl-removing) responsible for removal and (uridylytransferase) responsible for attachment of UMP to PII have been partially purified. These activities co-purify through a variety of procedures, including hydrophobic chromatography, and are stabilized by high ionic strength buffers. Whereas Mn2+ alone supports only uridylyl-removing activity, ATP, alpha-ketoglutarate, and Mg2+ support both uridylyl-removing and uridylyltransferase activities.     

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

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.     

Inactivation of glutamine synthetase by adenylylation in intact cells of E. coli

Summary The adenine pool of a purineless mutant of E. coli was radioactively labelled by short incubation with ¹⁴C-adenine.The glutamine synthetase was inactivated in vivo by incubation of the cell suspension with 2x10^-3 M NH₄ ⁺ for 2 min. The inactivated glutamine synthetase was extracted from the cells and purified 20-fold.Incubation of the purified glutamine synthetase with phosphodiesterase regenerated the biosynthetic activity of the enzyme paralleled by the liberation of ¹⁴C-adenine and ¹⁴C-adenosine. ¹⁴C-adenine and ¹⁴C-adenosine were also obtained when inactivated glutamine synthetase, prepared in vitro by use of ¹⁴C-ATP and purified adenylylating enzyme, was incubated with phosphodiesterase under the same conditions.The similar liberation of adenine derivatives by phosphodiesterase from glutamine synthetase inactivated in a cell-free system as well as in intact cells, demonstrates that in both cases the inactivation consists in an adenylylation of the enzyme.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH₄⁺ is in equilibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic activity of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in γ-glutamyl transferase assays containing 60 mM Mg²⁺. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of γ-glutamyl transferase activities without and with addition of 60 mM Mg²⁺.Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH₄⁺, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH₄⁺ produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Escherichia coli glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49): kinetic characterization of regulation by PII, PII-UMP, glutamine, and alpha-ketoglutarate

Glutamine synthetase adenylyltranferase (ATase, EC 2.7.7.49) catalyzes the adenylylation and deadenylylation of glutamine synthetase (GS), regulating GS activity. The adenylyltransferase (AT) reaction is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by the uridylylated form of PII, PII-UMP. Conversely, the adenylyl-removing (AR) reaction is activated by PII-UMP and is inhibited by glutamine and by PII. Both AT and AR reactions are regulated by alpha-ketoglutarate, which binds to PII and PII-UMP. Here, we present a kinetic analysis of the AT and AR activities and their regulation. Both AT and AR reactions used a sequential mechanism of rapid equilibrium random binding of substrates and products. Activators and inhibitors had little effect on the binding of substrates, instead exerting their effects on catalysis. Our results were consistent with PII, PII-UMP, and glutamine shifting the enzyme among at least six different enzyme forms, two of which were inactive, one of which exhibited AR activity, and three of which exhibited AT activity. In addition to a site for glutamine, the enzyme appeared to contain two distinct sites for PII and PII-UMP. The PII, PII-UMP, and glutamine sites were in communication so that the apparent activation and inhibition constants for regulators depended upon each other. The binding of PII was favored by glutamine and its level reduced by PII-UMP, whereas glutamine and PII-UMP competed for the enzyme. alpha-Ketoglutarate, which acts exclusively through its binding to PII and PII-UMP, did not alter the binding of PII or PII-UMP to the enzyme. Rather, alpha-ketoglutarate dramatically affected the extent of activation or inhibition of the enzyme by PII or PII-UMP. A working hypothesis for the regulation of the AT and AR activities, consistent with all data, is presented.     

Glutamine synthetase adenylyltransferase from Escherichia coli: purification and physical and chemical properties.

The glutamine synthetase adenylyltransferase (EC 2.7.7.42), WHIch catalyzes the adenylylation and deadenylylation of glutamine synthetase in E. coli, has been stabilized and purified 2200-fold to apparent homogeneity. Sedimentation and electrophoresis studies show that the native enzyme is a single polypeptide chain of 115,000 +/- 5000 molecular weight with an isoelectric pH (PL) OF 4.98, a sedimentation coefficient (S20.w0) of 5.6S, and a molar frictional coefficient (f/f0) of 1.52. An alpha-helical content of approximately equal to 25% and approximately equal to 28% beta-pleated sheet and approximately equal to 47% random coil structures were estimated from circular dichroism measurements. The amino acid composition of the protein has been determined. The intrinsic tryptophanyl residue flourescence of adenylyltransferase is two fold greater than that of L-tryptophan; this property has been used to monitor ligand-induced conformational changes in the enzyme. Activators of the adenylylation reaction (ATP, L-glutamine, or the E. coli PII regulatory protein) produced an enhancement of fluorescence; alpha-ketoglutarate, an inhibitor of adenylylation and an activator of deadenulylation, caused a net decrease in fluorescence. The adenylytransferase has separate interaction sites for L-glutamine and the regulatory PII protein.     

Activity of nitrogenase and glutamine synthetase in relation to availability of oxygen in continuous cultures of a strain of cowpea Rhizobium sp. supplied with excess ammonium.

In samples from nitrogen-fixing continuous cultures of strain CB756 of the cowpea type rhizobia (Rhizobium sp.), newly fixed NH+4 is in equiblibrium with the medium, from where it is assimilated by the glutamine synthetase/glutamate synthase pathway. In samples from steady state cultures with different degrees of oxygen-limitation, nitrogenase activity was positively correlated with the biosynthetic of glutamine synthetase in cell free extracts. Also, activities in biosynthetic assays were positively correlated with activities in gamma-glutamyl transferase assays containing 60 mM Mg2+. Relative adenylylation of glutamine synthetase was conveniently measured in cell free extracts as the ratio of gamma-glutamyl transferase activities without and with addition of 60 mM Mg2+. Automatic control of oxygen supply was used to facilitate the study of transitions between steady-state continuous cultures with high and low nitrogenase activities. Adenylylation of glutamine synthetase and repression of nitrogenase activity in the presence of excess NH+4, were masked when oxygen strongly limited culture yield. Partial relief of the limitation in cultures supplied with 10 mM NH+4 produced early decline in nitrogenase activity and increase in relative adenylylation of glutamine synthetase. Decreased oxygen supply produced a rapid decline in relative adenylylation, followed by increased nitrogenase activity, supporting the concept that control of nitrogenase synthesis is modulated by glutamine synthetase adenylylation in these bacteria.     

Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria.

The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.     

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.     

Probing interactions of the homotrimeric PII signal transduction protein with its receptors by use of PII heterotrimers formed in vitro from wild-type and mutant subunits.

The homotrimeric PII signal transduction protein of Escherichia coli interacts with two small-molecule effectors, 2-ketoglutarate and ATP, regulates two protein receptors, the kinase/phosphatase nitrogen regulator II (NRII) and the glutamine synthetase (GS) adenylyltransferase (ATase), and is subject to reversible uridylylation, catalyzed by the uridylyltransferase/uridylyl-removing enzyme (UTase/UR). The site of PII uridylylation, Y51, is located at the apex of the solvent-exposed T-loop (E. Cheah, P. D. Carr, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Structure 2:981-990, 1994), and an internally truncated PII lacking residues 47 to 53 formed trimers that bound the small-molecule effectors but were unable to be uridylylated or activate NRII and ATase (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179:4342-4353, 1997). We investigated the ability of heterotrimers containing delta47-53 and wild-type subunits to become uridylylated and activate NRII and ATase. Heterotrimers were formed by denaturation and renaturation of protein mixtures; when such mixtures contained a fivefold excess of A47-53 subunits, the wild-type subunits were mostly redistributed into trimers containing one wild-type subunit and two mutant subunits. The resulting population of trimers was uridylylated and deuridylylated by UTase/UR, stimulated the phosphatase activity of NRII, and stimulated adenylylation of GS by ATase. In all except the ATase interaction, the activity of the hybrid trimers was greater than expected based on the number of wild-type subunits present. These results indicate that a single T-loop region within a trimer is sufficient for the productive interaction of PII with its protein receptors. We also formed heterotrimers containing wild-type subunits and subunits containing the G89A alteration (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schefke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). The G89A mutant form of PII does not bind the small-molecule effectors, does not interact with UTase or with NRII, and interacts poorly with ATase. Heterotrimers formed with a 10/1 starting ratio of G89A to wild-type subunits interacted with UTase/UR and ATase to a lesser extent than expected based on the number of wild-type subunits present but activated NRII slightly better than expected based on the number of wild-type subunits present. Thus, intersubunit interactions within the PII trimer can adversely affect the activity of wild-type subunits and may affect the interactions with the different receptors in a variable way. Finally, we formed heterotrimers containing delta47-53 and G89A mutant subunits. These heterotrimers were not uridylylated, did not interact with NRII, and interacted with the ATase only to the extent expected based on the number of G89A subunits present. Thus, the G89A subunits, which contain an intact T-loop region, were not "repaired" by inclusion in heterotrimers along with delta47-53 subunits.     

Structure-function analysis of glutamine synthetase adenylyltransferase (ATase, EC 2.7.7.49) of Escherichia coli

Glutamine synthetase adenylyltransferase (ATase) regulates the activity of glutamine synthetase by adenylylation and deadenylylation in response to signals of nitrogen and carbon status: glutamine, alpha-ketoglutarate, and the uridylylated and unmodified forms of the PII signal transduction protein. ATase consists of two conserved nucleotidyltransferase (NT) domains linked by a central region of approximately 200 amino acids. Here, we study the activities and regulation of mutated and truncated forms of ATase. Our results indicate the following. (i) The N-terminal NT domain contained the adenylyl-removing (AR) active site, and the C-terminal NT domain contained the adenylyltransferase (AT) active site. (ii) The enzyme contained a glutamine binding site, and glutamine increased the affinity for PII. (iii) The enzyme appeared to contain multiple sites for the binding of PII and PII-UMP. (iv) Truncated versions of ATase missing the C-terminal (NT) domain lacked both AT and AR activity, suggesting a role for the C-terminal NT domain in both activities. (v) The purified C-terminal NT domain and larger polypeptides containing this domain had significant basal AT activity, which was stimulated by glutamine. These polypeptides were indifferent to PII and PII-UMP, or their ATase activity was inhibited by either PII or PII-UMP. (vi) Certain point mutations in the central region or an internal deletion removing most of this part of the protein eliminated the AR activity and eliminated activation of the AT activity by PII, while not eliminating the binding of PII or PII-UMP. That is, these mutations in the central region appeared to destroy the communication between the PII and PII-UMP binding sites and the AT and AR active sites. (vii) Certain mutations in the central region of ATase appeared to dramatically improve the binding of glutamine to the enzyme. (viii) While the isolated AT and AR domains of ATase bound poorly to PII and PII-UMP, these domains bound PII and PII-UMP significantly better when linked to the central region of ATase. Together, our results indicate a highly coordinated enzyme, in which the AT and AR domains participate in each other's regulation and distant regulatory sites are in communication with each other. A model for the regulation of ATase by glutamine, PII, and PII-UMP consistent with all data is presented.