Adenylylation State Of Glutamine Synthetase Research Articles

REGULATION OF NITROGEN FIXATION BY THE METABOLISM OF CARBON COMPOUNDS

ABSTRACT Nitrogen fixation in Rhodopseudomonas capsulata was subject to regulation by carbon metabolism. Citric acid cycle intermediates participated in this regulation, either directly or through accumulation of unknown derivatives of their metabolism. The carboxylic acids appeared to inhibit the A (or active) form of nitrogenase that predominates in nitrogen-starved cells. In exponentially growing cells in lactate-glutamate medium, where the R (or regulated) form of nitrogenase predominates, the citric acid cycle intermediates inhibited C2H2 reduction activity by only 10–35%. Citrate and isocitrate were the most potent inhibitors, though full inhibition was not achieved before periods of one to several hours after their addition. The inhibitory effect of the 6-carbon carboxylic acids was not due to repression of enzyme synthesis, nor was it accompanied by changes in the adenylylation state of glutamine synthetase. Oxaloacetate, the immediate precursor of citrate, also caused inhibition of C2H2 reduction...

The multifarious short‐term regulation of ammonium assimilation of Escherichia coli: dissection using an in silico replica

Ammonium assimilation in Escherichia coli is regulated through multiple mechanisms (metabolic, signal transduction leading to covalent modification, transcription, and translation), which (in-)directly affect the activities of its two ammonium-assimilating enzymes, i.e. glutamine synthetase (GS) and glutamate dehydrogenase (GDH). Much is known about the kinetic properties of the components of the regulatory network that these enzymes are part of, but the ways in which, and the extents to which the network leads to subtle and quasi-intelligent regulation are unappreciated. To determine whether our present knowledge of the interactions between and the kinetic properties of the components of this network is complete - to the extent that when integrated in a kinetic model it suffices to calculate observed physiological behaviour - we now construct a kinetic model of this network, based on all of the kinetic data on the components that is available in the literature. We use this model to analyse regulation of ammonium assimilation at various carbon statuses for cells that have adapted to low and high ammonium concentrations. We show how a sudden increase in ammonium availability brings about a rapid redirection of the ammonium assimilation flux from GS/glutamate synthase (GOGAT) to GDH. The extent of redistribution depends on the nitrogen and carbon status of the cell. We develop a method to quantify the relative importance of the various regulators in the network. We find the importance is shared among regulators. We confirm that the adenylylation state of GS is the major regulator but that a total of 40% of the regulation is mediated by ADP (22%), glutamate (10%), glutamine (7%) and ATP (1%). The total steady-state ammonium assimilation flux is remarkably robust against changes in the ammonium concentration, but the fluxes through GS and GDH are completely nonrobust. Gene expression of GOGAT above a threshold value makes expression of GS under ammonium-limited conditions, and of GDH under glucose-limited conditions, sufficient for ammonium assimilation.

The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state.

The regulation of Escherichia coli glutamine synthetase (GS) by reversible adenylylation has provided one of the classical paradigms for signal transduction by cyclic cascades. Yet, many mechanistic features of this regulation remain to be elucidated. We examined the regulation of GS adenylylation state in a reconstituted system containing GS, adenylyltransferase (ATase), the PII signal transduction protein that controls ATase, and the uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which has a role in regulating PII. In this reconstituted bicyclic cascade system, the adenylylation state of GS was regulated reciprocally by the small molecule effectors 2-ketoglutarate and glutamine at physiological effector concentrations. By examination of the individual regulatory monocycles and comparison to the bicyclic system and existing data, we could deduce that the only sensors of 2-ketoglutarate were PII and PII-UMP. At physiological conditions, we observed that the main role of 2-ketoglutarate in bringing about the deadenylylation of GS was to inhibit GS adenylylation, and this was due to the allosteric regulation of PII activity. Glutamine acted as an allosteric regulator of both ATase and UTase/UR. We also compared the regulation of GS adenylylation state to the regulation of phosphorylation state of the transcription factor NRI (NtrC) in a reconstituted bicyclic system containing NRI, the bifunctional kinase/phosphatase NRII (NtrB), PII, and the UTase/UR. This comparison indicated that, at a fixed 2-ketoglutarate concentration, the regulation of GS adenylylation state by glutamine was sharper and occurred at a higher concentration than did the regulation of NRI phosphorylation. The possible biological implications of this regulatory arrangement are discussed.

Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli.

Two structurally similar but functionally distinct PII-like proteins, PII and GlnK, regulate nitrogen assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphorylation state of the transcriptional activator NRI-P (NtrC-P, the phosphorylated form of the glnG (ntrC) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA, which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on nitrogen-rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK*, were obtained as suppressors of the Ntr- phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRI-P during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

Adenylylation state of glutamine synthetase and permeability properties of Pseudomonas fluorescens

The state of adenylylation, n̄, 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 NH 4 + low dicarboxylic acid substrate), the value of n̄ falls rapidly from 10 to 1.0 during prolonged incubation in the stationary phase, whereas the value of n̄ 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 α-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 α-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 NH 4 + 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.

Ammonia and light effect on nitrogenase activity in nitrogen-limited continuous cultures of Rhodopseudomonas capsulata. Role of glutamine synthetase

Nitrogen-limited continuous cultures of Rhodopseudomonas capsulata were used to investigate some aspects of the regulation of nitrogenase activity. The role of glutamine synthetase (GS) in this regulation was examined by measuring changes of its adenylylation state when the light intensity and the nitrogen source were varied. Maximal nitrogenase activity was observed at a dilution rate corresponding to about one third of the maximum specific growth rate (μmax), both in ammonia- and in glutamate-limited cultures. At higher dilution rates, both GS and nitrogenase were inactivated by ammonia. Determination of the kinetics of inhibition of both enzymes indicated that the degree of inactivation of nitrogenase and the adenylylation state of GS were not closely related. Increase of light intensity stimulated nitrogenase activity dramatically. Conversely, a shift-down in light intensity to a limiting value resulted in a decrease of nitrogenase activity suggesting that synthesis was inhibited. On the other hand, the adenylylation state of glutamine synthetase appeared to be unaffected by changes in light intensity, indicating that GS is probably not involved in the regulation of nitrogenase expression by light.

Regulation and biochemical characterization of the glutamine synthetase of Azotobacter vinelandii

We have investigated the regulation of the activity and synthesis of the glutamine synthetase ( lglutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) of Azotobacter vinelandii. Synthesis of the enzyme was not repressed by NH + 4 and/or a number of amino acids in the growth medium; however, biosynthetic activity was rapidly lost through adenylylation in response to ammonium ion. The enzyme could be prepared as a ‘relaxed’, divalent-cation-free form which was catalytically inactive. The ‘taut’, active form could be restored with 1–5 mM Mg 2+, Mn 2+, Ca 2+ or CO 2+ and taut-vs.-relaxed difference spectra unique to each divalent cation were generated. Mg 2+ and CO O2+ each supported biosynthetic catalysis, but with different substrate K m and V max values. l-Alanine, glycine and l-aspartate were the most potent of several inhibitors of the biosynthetic and the γ-glutamyl transferase activities; only aspartate and AMP behaved differentially toward glutamine synthetase adenylylation state: the more highly adenylylated enzyme was more severely affected. Any two of alanine, glycine or AMP showed cumulative inhibition, while the inhibitory effects of groups of three effectors were not cumulative. The Co 2+ -supported biosynthetic activity of A. vinelandii glutamine synthetase was markedly less sensitive to inhibition my glycine and alanine and was stimulated up to 50% by 1–10 mM aspartate.

Nitrogenase switch-off by ammonia in Rhodopseudomonas palustris: Loss under nitrogen deficiency and independence from the adenylylation state of glutamine synthetase

Nitrogenase activity in Rhodopseudomonas palustris is subject to a rapid switch-off in response to exogenous ammonia. When cells were grown on limiting nitrogen and eventually became nitrogen deficient, nitrogenase synthesis was fully derepressed but the enzyme was insensitive to ammonia. The transformation of ammonia-sensitive to ammonia-insensitive cells was a slow, but fully reversible process. The switch-off effect in ammonia-sensitive cells paralleled changes in the adenylylation state of glutamine synthetase. Ammonia-insensitive cells, however, showed similar changes in glutamine synthetase activity although nitrogenase activity was unaffected. We conclude that nitrogenase regulation and adenylylation of glutamine synthetase are independent processes, at least under conditions of nitrogen deficiency.

Changes in the regulatory form of Rhodospirillum rubrum nitrogenase as influenced by nutritional and environmental factors.

The photosynthetic bacterium Rhodospirillum rubrum regulates the activity of its nitrogenase (N2ase) by interconverting the enzyme into three distinct enzymatic species: N2ase A (a fully active form) and two regulatory forms, N2ase Ractive and N2ase Rinactive. N2ase R is distinguished from N2ase A in vitro by the requirement of its Fe protein for activation by a Mn2+-dependent activating factor. N2ase is converted from the A to the R form in response to certain environmental factors such as carbon starvation, depletion of intracellular adenosine triphosphate, or the addition of NH4+ (or glutamate) to a culture of N-starved cells. The rapid inhibition of R. rubrum N2ase in vivo by NH4+ was shown to result from the conversion of N2ase A to N2ase Rinactive. On depletion of NH4+ from the culture, whole-cell N2ase activity returned; however, the enzyme remained in the R form. Unlike the effect of NH4+, adding glutamate to cells containing N2ase A did not inhibit in vivo activity, but converted the enzyme to the R form (N2ase Ractive). Although glutamate-induced N2ase R formation was much slower than the NH4+-induced reaction, it occurred in the presence of rifampin, indicating that de novo protein synthesis was not involved. This suggested that N2ase R was formed by a modification of N2ase A. Although glutamine synthetase in involved in the conversion of N2ase A to R, the adenylylation state of glutamine synthetase appears not to be involved in regulating this nitrogenase reaction.

Relation between the adenylylation state of glutamine synthetase and the expression of other genes involved in nitrogen metabolism.

We have partially characterized the biochemical parameters of glutamine synthetase from Klebsiella pneumoniae and have shown that the differential affinity of adenylylated and unadenylylated glutamine synthetase for adenosine diphosphate provides a convenient means of determining the adenylylation state. Using this assay procedure, we examined the relationship between the adenylylation state and the expression of other genes involved in nitrogen assimilation. We observed no correlation between the adenylylation state and the expression of histidase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, and urease in aerobic cultures.

Biochemical parameters of glutamine synthetase from Klebsiella aerogenes.

The glutamine synthetase (GS) from Klebsiella aerogenes is similar to that from Escherichia coli in several respects: (i) it is repressed by high levels of ammonia in the growth medium; (ii) its biosynthetic activity is greatly reduced by adenylylation; and (iii) adenylylation lowers the pH optimum and alters the response of the enzymes to various inhibitors in the gamma-glutamyl transferase (gammaGT) assay. There are, however, several important differences: (i) the isoactivity point for the adenylylated and non-adenylylated forms in the gammaGT assay occurs at pH 7.55 in K. aerogenes and at pH 7.15 in E. coli; (ii) the non-adenylylated form of the GS from K. aerogenes is stimulated by 60 mM MgCl2 in the gammaGT assay at pH 7.15. A biosynthetic reaction assay that correlates well with number of non-adenylylated enzyme subunits, as determined by the method of Mg2+ inhibition of the gammaGT assay, is described. Finally, we have found that it is necessary to use special methods to harvest growing cells to prevent changes in the adenylylation state of GS from occurring during harvesting.