Regulation and properties of the glutamine synthetase purified from Photobacterium phosphoreum

Glutamine synthetase from a marine enterobacterium, Photobacterium phosphoreum, was purified to homogeneity from cells grown in glycerol-yeast extract medium. The purified enzyme had a molecular weight of approximately 670,000 and a subunit size of 56,000, i.e. larger than that of the enzyme from E. coli. Regulation of the glutamine synthetase activity by adenylylation/deadenylylation was demonstrated on snake venom phosphodiesterase treatment. The state of adenylylation appeared to influence both the biosynthetic and gamma-glutamyltransferase activities of P. phosphoreum glutamine synthetase similar to in the case of the E. coli enzyme. The enzyme activity was controlled by adenylylation and possibly in combination with feedback inhibition by alanine, serine, and glycine, metabolites which are especially effective in inhibiting P. phosphoreum glutamine synthetase. When either Mn2+ or Mg2+ was added to the relaxed (divalent cation-free) enzyme, similar UV-difference spectra were obtained for the enzyme, indicating that the conformational states induced by these cations were also similar. The profile of these spectra varied from those published for E. coli, and three peaks were four 1 at 282.5, 288.5, and 298 nm.     

Purification and properties of Azotobacter vinelandii glutamine synthetase.

A procedure is described for the purification of glutamine synthetase from the nitrogen-fixing organism Azotobacter vinelandii. Electron micrographs of the enzyme reveal a dodecameric arrangement of its subunits in two superimposed hexagonal rings similar to the glutamine synthetase of Escherichia coli. Disc eleetrophoresis in the presence of sodium dodecyl sulfate and sedimentation studies show a subunit molecular weight of 56,500 and a sedimentation coefficient (s_20,w) of the native enzyme of 20.0 S. Like the E. coli enzyme, the glutamine synthetase of A. vinelandii is regulated by adenylylation/deadenylylation. This finding was derived from (a) studies on the effect of snake venom phosphodiesterase treatment on the catalytic and spectral properties of enzyme isolated from cells grown on a nitrogen-rich medium, (b) the identification of the AMP released by the phosphodiesterase by thin-layer chromatography, (c) the selective precipitation of adenylylated enzyme with antibodies directed against adenylylated bovine serum albumin, and (d) the in vitro incorporation of radioactivity from [¹⁴C]ATP into deadenylylated enzyme in the presence of either crude extract from A. vinelandii or partially purified adenylyl transferase from E. coli. The state of adenylylation appears to have a similar influence on the catalytic properties of A. vinelandii glutamine synthetase as on those of the E. coli enzyme, with the exception that the deadenylylated form of the A. vinelandii glutamine synthetase is almost inactive in the Mn-dependent transferase reaction.     

Adenylylation and catalytic properties of Mycobacterium tuberculosis glutamine synthetase expressed in Escherichia coli versus mycobacteria

Bacterial glutamine synthetases (GSs) are complex dodecameric oligomers that play a critical role in nitrogen metabolism, converting ammonia and glutamate to glutamine. Recently published reports suggest that GS from Mycobacterium tuberculosis (MTb) may be a therapeutic target (Harth, G., and Horwitz, M. A. (2003) Infect. Immun. 71, 456-464). In some bacteria, GS is regulated via adenylylation of some or all of the subunits within the aggregate; catalytic activity is inversely proportional to the extent of adenylylation. The adenylylation and deadenylylation of GS are catalyzed by adenylyl transferase (ATase). Here, we demonstrate via electrospray ionization mass spectrometry that GS from pathogenic M. tuberculosis is adenylylated by the Escherichia coli ATase. The adenylyl group can be hydrolyzed by snake venom phosphodiesterase to afford the unmodified enzyme. The site of adenylylation of MTb GS by the E. coli ATase is Tyr-406, as indicated by the lack of adenylylation of the Y406F mutant, and, as expected, is based on amino acid sequence alignments. Using electrospray ionization mass spectroscopy methodology, we found that GS is not adenylylated when obtained directly from MTb cultures that are not supplemented with glutamine. Under these conditions, the highly related but non-pathogenic Mycobacterium bovis BCG yields partially ( approximately 25%) adenylylated enzyme. Upon the addition of glutamine to the cultures, the MTb GS becomes significantly adenylylated ( approximately 30%), whereas the adenylylation of M. bovis BCG GS does not change. Collectively, the results demonstrate that MTb GS is a substrate for E. coli ATase, but only low adenylylation states are accessible. This parallels the low adenylylation states observed for GS from mycobacteria and suggests the intriguing possibility that adenylylation in the pathogenic versus non-pathogenic mycobacteria is differentially regulated.     

Purification of PII and PII-UMP and In Vitro Studies of Regulation of Glutamine Synthetase in Rhodospirillum rubrum

The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography. The uridylylated form of the P(II) protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and alpha-ketoglutarate were studied in extracts from R. rubrum grown under different conditions. P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by alpha-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. coli system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.     

No evidence of covalent modification of glutamine synthetase in the thermophilic phototropic bacterium Chloroflexus aurantiacus

Abstract Regulation of glutamine synthetase (GS) in the thermophilic green phototrophic bacterium, Chloroflexus aurantiacus , was studied. The enzyme was partially purified from cells grown photosynthetically in media with limiting (1 mM) or non-limiting (10 mM) NH⁺₄-concentrations. GS preparations from both cell types were indistinguishable in respect to pH-optimum of GS-transferase activity, sensitivity to feedback modifiers (AMP, L-alanine, glycine) and lack of Mg-inhibition of transferase activity. In contrast to results obtained with a GS preparation from the facultatively phototrophic bacterium, Rhodopseudomonas sphaeroides , the catalytic properties of Chloroflexus GS did not change during incubation with snake venom phosphodiesterase suggesting the absence of in vivo regulation of Chloroflexus GS by adenylylation/deadenylylation.     

In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803.

Glutamine synthetase from Synechocystis sp. strain PCC 6803 is inactivated by ammonium addition to cells growing with nitrate as the nitrogen source. The enzyme can be reactivated in vitro by different methods such as alkaline phosphatase treatment, but not phosphodiesterase, by raising the pH of the crude extract to values higher than 8, by increasing the ionic strength of the cell-free extract, or by preincubation with organic solvents, such as 2-propanol and ethanol. These results suggest that the loss of glutamine synthetase activity promoted by ammonium involves the non-covalent binding of a phosphorylated compound to the enzyme and support previous results that rule out the existence of an adenylylation/deadenylylation system functioning in the regulation of cyanobacterial glutamine synthetase.     

Regulation of glutamine synthetase activity by phosphorylation instead of by adenylylation

o-Phosphotyrosyl glutamine synthetase (P-GS) was isolated from highly adenylated glutamine synthetase (AMP-GS) purified from Mycobacterium phlei, by treatment with micrococcal nuclease. The physical characteristics of P-GS were quite similar to those of AMP-GS except for the UV-absorption spectrum. In either Mg2+- or Mn2+-dependent biosynthetic reactions, the kinetic properties, such as optimum pH, Vmax, and apparent Km for each of three substrates of P-GS, were found to be in good agreement with those of AMP-GS. The biosynthetic activity of P-GS was markedly increased after treatment with alkaline phosphatase similarly as in the deadenylylation of AMP-GS by snake venom phosphodiesterase treatment. These results revealed that repression of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue, without recourse to adenylylation.     

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.     

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.     

The glutamine synthetase-glutamate synthase system in Rhodopseudomonas sphaeroides

The phototrophic purple bacterium Rhodopseudomonas sphaeroides, strain 2R, can assimilate ammonium by means of glutamine synthetase and glutamate synthase. A higher activity of glutamine synthetase is displayed by cells grown in the medium with glutamate or in the atmosphere of molecular nitrogen. The activity of glutamate synthase also rises when cells grow in the atmosphere of N2. However, in contrast to glutamine synthetase, the activity of glutamate synthase does not decrease in the presence of considerable NH4+ amounts. The glutamine synthetase of R. sphaeroides is modified by adenylylation/deadenylylation. In the presence of nitrogenase in R. sphaeroides, the glutamine synthetase is found mainly in the deadenylylation state. Methionine sulfone, an inhibitor of glutamine synthetase, partly restores the activity of nitrogenase in the presence of ammonium, and prevents adenylylation of glutamine synthetase.     

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.     

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.     

Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations

Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.     

Physical and chemical characterization of glutamine synthetase purified from Mycobacterium phlei

Glutamine synthetase (L-glutamate: ammonia ligase [ADP forming]) [EC 6.3.1.2] has been purified from a Gram-positive, acid-fast bacterium, Mycobacterium phlei, by simple procedures with 57% recovery. The enzyme resembled that from Mycobacterium smegmatis in the subunit size (56,000), molecular weight (670,000), amino acid composition, the amino acid sequence of the NH2-terminal, and the secondary structure. The enzyme activity was regulated by adenylylation of each subunit in the dodecameric molecule. M. phlei glutamine synthetase possesses two useful characteristics: high thermostability and resistance to protease digestion. The enzyme was not inactivated on exposure to 60 degrees C for 2 h or 37 degrees C for 72 h, or after incubation with 1% trypsin or chymotrypsin at 37 degrees C for 12 h, pH 7.8. With saturating substrate levels, the Arrhenius plot was nonlinear and concave downward with an intersection point at 45 degrees C, and the activation energies were calculated to be 3.2 and 9.6 cal/mol from the slopes. The specific activity of the highly adenylylated enzyme (E10.7) was remarkably lower than that of the slightly adenylylated enzyme (E2.5); however, both enzymes show similar profiles of the Arrhenius plot. These results indicate that the adenylylation of the enzyme does not affect its activation energies.     

The domains carrying the opposing activities in adenylyltransferase are separated by a central regulatory domain

Adenylyltransferase is a bifunctional enzyme that controls the enzymatic activity of dodecameric glutamine synthetase in Escherichia coli by reversible adenylylation and deadenylylation. Previous studies showed that the two similar but chemically distinct reactions are carried out by separate domains within adenylyltransferase. The N-terminal domain carries the deadenylylation activity, and the C-terminal domain carries the adenylylation activity [Jaggi R, van Heeswijk WC, Westerhoff HV, Ollis DL & Vasudevan SG (1997) EMBO J16, 5562-5571]. In this study, we further map the domain junctions of adenylyltransferase on the basis of solubility and enzymatic analysis of truncation constructs, and show for the first time that adenylyltransferase has three domains: the two activity domains and a central, probably regulatory (R), domain connected by interdomain Q-linkers (N-Q1-R-Q2-C). The various constructs, which have the opposing domain and or central domain removed, all retain their activity in the absence of their respective nitrogen status indicator, i.e. PII or PII-UMP. A panel of mAbs to adenylyltransferase was used to demonstrate that the cellular nitrogen status indicators, PII and PII-UMP, probably bind in the central regulatory domain to stimulate the adenylylation and deadenylylation reactions, respectively. In the light of these results, intramolecular signaling within adenylyltransferase is discussed.     

Metabolite regulation of the state of adenylylation of glutamine synthetase

When glutamine synthetase is incubated in a mixture containing adenylyltrans-ferase, the regulatory protein (P_II) and several effectors, including ATP, UTP, P_i, α-ketoglutarate, glutamine, and Mg²⁺ and/or Mn²⁺, it ultimately assumes a constant state of adenylylation. The final state of adenylylation (i.e., the number of adenylylated subunits per mole of enzyme) can vary from 0 to 12 and is specified by the concentrations and ratios of the various effectors and by the extent of uridylylation of P_II (i.e., the P_IIA:P_IID ratio). Under otherwise identical conditions, increasing the concentrations of either UTP, P_i, α-ketoglutarate, Mn²⁺, or P_IID decreases the state of adenylylation finally reached, whereas increasing the concentrations of either glutamine, ATP, or Pua increases the final state of adenylylation. The final state of adenylylation is independent of the concentrations of glutamine synthetase, adenylyltransferase, and P_II (but not of the P_IIA:P_IIDratio), and also of the initial average state of adenylylation of glutamine synthetase. Various lines of evidence show that the final state of adenylylation represents a dynamic steady state in which the rates of adenylylation and deadenylylation of glutamine synthetase are equal. It is concluded that the regulation of glutamine synthetase activity by the adenylylation mechanism utilizes a significant amount of ATP energy, but this amount is less than 0.1% that utilized directly by the glutamine synthetase in the synthesis of glutamine.     

Heterotrimerization of PII-like signalling proteins: implications for PII-mediated signal transduction systems

PII-like signalling molecules are trimeric proteins composed of 12-13 kDa polypeptides encoded by the glnB gene family. Heterologous expression of a cyanobacterial glnB gene in Escherichia coli leads to an inactivation of E. coli's own PII signalling system. In the present work, we show that this effect is caused by the formation of functionally inactive heterotrimers between the cyanobacterial glnB gene product and the E. coli PII paralogues GlnB and GlnK. This led to the discovery that GlnK and GlnB of E. coli also form heterotrimers with each other. The influence of the oligomerization partner on the function of the single subunit was studied using heterotrimerization with the Synechococcus PII protein. Uridylylation of GlnB and GlnK was less efficient but still possible within these heterotrimers. In contrast, the ability of GlnB-UMP to stimulate the adenylyl-removing activity of GlnE (glutamine synthetase adenylyltransferase/removase) was almost completely abolished, confirming that rapid deadenylylation of glutamine synthetase upon nitrogen stepdown requires functional homotrimeric GlnB protein. Remarkably, however, rapid adenylylation of glutamine synthetase upon exposing nitrogen-starved cells to ammonium was shown to occur in the absence of a functional GlnB/GlnK signalling system as efficiently as in its presence.     

Adenylylation state of glutamine synthetase and permeability properties of Pseudomonas fluorescens

The state of adenylylation, n, of glutamine synthetase (GS) in Pseudomonas fluorescens has been determined as a function of growth conditions. Compared to the behavior of Escherichia coli, atypical responses to either carbon or nitrogen starvation were observed when P. fluorescens was grown with either succinate, malate, or fumarate as the sole source of carbon and energy. Under conditions of carbon starvation (high NH4+, low dicarboxylic acid substrate), the value of n falls rapidly from 10 to 1.0 during prolonged incubation in the stationary phase, whereas the value of n is unexpectedly high (ca. 10) in extracts of nitrogen-starved cells. These abnormal responses are attributable to particular permeability properties of P. fluorescens cells compared to E. coli. The unusual changes in nitrogen-starved cells are related to the release of alpha-ketoglutarate by such cells during incubation or washing procedures. These changes can be prevented by the addition of cetyltrimethylammonium bromide (CTAB) to the cultures 5 min prior to harvesting the cells, or by freezing the cell pellets just after centrifugation and sonication within 3 min of suspension in buffer, or by suspending freshly harvested cells in buffer containing alpha-ketoglutarate and orthophosphate (i.e., effectors that favor deadenylylation of glutamine synthetase). The abnormal changes which occur during carbon starvation in the presence of excess NH4+ can be prevented by addition of ATP and glutamine to the buffer in which the freshly harvested cells are suspended prior to sonication. The results suggest that during the stationary phase of growth on succinate, fumarate, or malate (but not on glucose), the cellular membrane becomes permeable to small molecules that regulate the adenylylation cascade, and indeed, it was observed that such whole cells expressed, without any chemical or physical treatment, more than 50% of the glutamine synthetase activity they contained. Such cells may be useful in studies to examine the effects of multiple metabolites on the regulation of glutamine synthetase adenylylation in situ.     

Evidence for adenylylation/deadenylylation control of the glutamine synthetases of Rhodospirillum tenue and Rhodocyclus purpureus

The phylogenetically related phototrophic bacteria Rhodospirillum tenue and Rhodocyclus purpureus modulate activity of their glutamine synthetases by adenylylation/deadenylylation. Evidence for covalent modification includes the inhibitory effect of Mg²⁺ on the activity of glutamine synthetase extracted from cells of either species grown on excess ammonia, and the lack of Mg²⁺ inhibition of activity of the enzyme isolated from N₂-(R. tenue) or glutamine (R. purpureus)-grown cells. In addition, snake venom phosphodiesterase treatment of glutamine synthetase from either species grown on excess ammonia relieved Mg²⁺ inhibition of the enzyme (as measured via the -glutamyl transferase assay), and changed the cation specificity from Mn²⁺ to Mg²⁺ (in the biosynthetic assay).