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

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.     

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.     

Robustness in Glyoxylate Bypass Regulation

The glyoxylate bypass allows Escherichia coli to grow on carbon sources with only two carbons by bypassing the loss of carbons as CO₂ in the tricarboxylic acid cycle. The flux toward this bypass is regulated by the phosphorylation of the enzyme isocitrate dehydrogenase (IDH) by a bifunctional kinase–phosphatase called IDHKP. In this system, IDH activity has been found to be remarkably robust with respect to wide variations in the total IDH protein concentration. Here, we examine possible mechanisms to explain this robustness. Explanations in which IDHKP works simultaneously as a first-order kinase and as a zero-order phosphatase with a single IDH binding site are found to be inconsistent with robustness. Instead, we suggest a robust mechanism where both substrates bind the bifunctional enzyme to form a ternary complex.     

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.     

The deuridylylation activity of Herbaspirillum seropedicae GlnD protein is regulated by the glutamine:2-oxoglutarate ratio

The nitrogen metabolism of Proteobacteria is controlled by the general Ntr system in response to nitrogen quality and availability. The PII proteins play an important role in this system by modulating the cellular metabolism through physical interaction with protein partners. Herbaspirillum seropedicae, a nitrogen-fixing bacterium, has two PII proteins paralogues, GlnB and GlnK. The interaction of H. seropedicae PII proteins with its targets is regulated by allosteric ligands and by reversible post-translational uridylylation. Both uridylylation and deuridylylation reactions are catalyzed by the same bifunctional enzyme, GlnD. The mechanism of regulation of GlnD activity is still not fully understood. Here, we characterized the regulation of deuridylylation activity of H. seropedicae GlnD in vitro. To this purpose, fully modified PII proteins were submitted to kinetics analysis of its deuridylylation catalyzed by purified GlnD. The deuridylylation activity was strongly stimulated by glutamine and repressed by 2-oxoglutarate and this repression was strong enough to overcome the glutamine stimulus of enzymatic activity. We also constructed and analyzed a truncated version of GlnD, lacking the C-terminal regulatory ACT domains. The GlnDΔACT protein catalyzed the futile cycle of uridylylation and deuridylylation of PII, regardless of glutamine and 2-oxoglutarate levels. The results presented here suggest that GlnD can sense the glutamine:2-oxoglutarate ratio and confirm that the ACT domains of GlnD are the protein sensors of environment clues of nitrogen availability.     

Structure of the Adenylylation Domain of E. coli Glutamine Synthetase Adenylyl Transferase: Evidence for Gene Duplication and Evolution of a New Active Site

The X-ray structure of the C-terminal fragment, containing residues 449-946, of Escherichia coli glutamine synthetase adenylyl transferase (ATase) has been determined. ATase is part of the cascade that regulates the enzymatic activity of E. coli glutamine synthetase, a key component of the cell's machinery for the uptake of ammonia. It has two enzymatic activities, adenylyl removase (AR) and adenylyl transferase (AT), which are located in distinct catalytic domains that are separated by a regulatory (R) domain. We previously reported the three-dimensional structure of the AR domain (residues 1-440). The present structure contains both the R and AT domains. AR and AT share 24% sequence identity and also contain the β-polymerase motif that is characteristic of many nucleotidylyl transferase enzymes. The structures overlap with an rmsd of 2.4 Å when the superhelical R domain is omitted. A model for the complete ATase molecule is proposed, along with some refinements of domain boundaries. A rather more speculative model for the complex of ATase with glutamine synthetase and the nitrogen signal transduction protein PII is also presented.     

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.     

The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin.

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and 'half-maximal concentration' values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.     

Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique.

Ammonia-nitrogen-limited continuous cultures of Escherichia coli and Klebsiella aerogenes contain induced levels of glutamine synthetase that is deadenylyated (i.e., fully active). In the presence of excess ammonia or glutamate in glucose-limited cultures of E. coli, glutamine synthetase is repressed and adenylylated (inactive). The average state of adenylylation (n) is a linear function of the specific growth rate. At low specific growth rates, glutamine synthetase is adenylylated; as the specific growth rate increases, n decreases, approaching 0 to 2 at rapid growth rates. The average state of adenylylation correlates well with the intracellular concentrations and ratios of alpha-ketoglutarate and glutamine, which are key effectors in the adenylylation-deadenylylation systems. E. coli and K. aerogenes differ markedly in their growth yields, growth rates, and enzymatic composition during nitrogen limitation. The data suggest that, unlike K. aerogenes, E. coli W uses glutamate dehydrogenase to incorporate ammonia during nitrogen limitation. In E. coli, glutamate dehydrogenase is progressively induced during nitrogen limitation when mu (growth rate) approaches mumax. In contrast, in K. aerogenes glutamate dehydrogenase is repressed during nitrogen limitation, whereas glutamate synthase, an alternative supplier of glutamate to the cell, is induced. Data are presented that support the regulatory schemes proposed for the control of glutamine synthetase activity by induction-repression phenomena and adenylylation-deadenylylation reaction. We propose that the intracellular ratio of alpha-ketoglutarate to glutamine may be the most important physiological parameter in determining the activity of glutamine synthetase.     

Immunochemical evidence for glutamine-mediated degradation of glutamine synthetase in cultured Chinese hamster cells.

The specific activity of glutamine synthetase in cultured Chinese hamster cells is inversely related to the concentration of glutamine in the surrounding solution. Enzyme specific activity increases 8- to 10-fold when glutamine is removed from serum-free F12 growth media. The induction of glutamine synthetase activity occurs only after glutamine removal and not after the removal of other amino acids (methionine, leucine, or isoleucine). The analysis of the glutamine-mediated decrease in glutamine synthetase activity has been simplified by the finding that depression proceeds in nutrient-free buffered saline solution (141 mM NaCl, 5.4 mM KCl and 30 mM Tricine (pH 7.4). Under these conditions, 0.1 mM cyanide blocks glutamine-mediated depression. The cyanide inhibition is reversed by the addition of 1.0 mM glucose which suggests that ATP is required for depression. Glutamine-mediated depression is temperature-dependent, occurring between 25 and 45 degrees with an optimum rate at 37 degrees. Studies of the time course of induction and depression as a function of glutamine concentration suggest that glutamine regulates the rate at which the enzyme is either modified or degraded. We have employed an antibody prepared against homogeneous Chinese hamster liver glutamine synthetase to measure the amount of glutamine synthetase protein in extracts of cells containing induced or depressed levels of enzyme activity. A highly sensitive immunoprecipitation procedure enables quantitation of nanogram amounts of glutamine synthetase protein. Glutamine synthetase in cell extracts containing induced levels of enzyme activity possesses the same molecular specific activity (ratio of activity to antigenicity) as homogeneous Chinese hamster liver glutamine synthetase. The molecular specific activity of glutamine synthetase is almost the same in extracts of cells with depressed levels of enzyme obtained by growth for short (2 hours) and long (24 hours) times in the presence of glutamine. These data suggest that glutamine-mediated depression of glutamine synthetase results from degradation of enzyme molecules.     

Allosteric interactions and bifunctionality make the response of glutamine synthetase cascade system of Escherichia coli robust and ultrasensitive

Glutamine synthetase (GS) regulation in Escherichia coli by reversible covalent modification cycles is a prototype of signal transduction by enzyme cascades. Such enzyme cascades are known to exhibit ultrasensitive response to primary stimuli and act as signal integration systems. Here, we have quantified GS bicyclic cascade based on steady state analysis by evaluating Hill coefficient. We demonstrate that adenylylation of GS with glutamine as input is insensitive to total enzyme concentrations of GS, uridylyltransferase/uridylyl-removing enzyme, regulatory protein PII, and adenylyltransferase/adenylyl-removing enzyme. This robust response of GS adenylylation is also observed for change in system parameters. From numerical analyses, we show that the robust ultrasensitive response of bicyclic cascade is because of allosteric interactions of glutamine and 2-ketoglutarate, bifunctionality of converter enzymes, and closed loop bicyclic cascade structure. By system level quantification of the GS bicyclic cascade, we conclude that such a robust response may help the cell in adapting to different carbon and nitrogen status.     

Salmonella typhimurium LT-2 mutants with altered glutamine synthetase levels and amino acid uptake activities.

To determine whether Salmonella typhimurium has a nitrogen control response, we have examined the regulation of nitrogen utilization in two mutants with fivefold and threefold elevations in their glutamine synthetase activities. The mutants do not require glutamine for growth on glucose--ammonia medium but do have altered growth on other nitrogen sources. They grow better than an isogenic control on media containing arginine or asparate, but more slowly with proline or alanine as nitrogen sources. This unusual growth pattern is not due to altered regulation of the ammonia assimilatory enzymes, glutamate dehydrogenase and glutamate synthase, or to changes in the enzymes for aspartate degradation. However, transport for several amino acids may be affected. Measurement of amino acid uptake show that the mutants with high glutamine synthetase levels have increased rates for glutamine, arginine, aspartate, and lysine, but a decreased rate for proline. The relationship between glutamine synthetase levels and uptake was examined in two mutants with reduced, rather than increased, glutamine synthetase production. The uptake rates for glutamine and lysine were lower in these two glutamine auxotrophs than in the Gln+ controls. These results show a correlation between the glutamine synthetase levels and the uptake rates for several amino acids. In addition, the pleiotropic growth of the mutants with elevated glutamine synthetase activities suggests that a nitrogen control response exists for S. typhimurium and that it can be altered by mutations affecting glutamine synthetase regulation.     

高氨氮利用酵母菌的筛选及相关酶活性

【背景】蛋白饲料的缺乏,促进了蛋白含量高、安全性能好的酵母类单细胞蛋白的研究与应用。【目的】筛选氨氮利用能力强的菌株,为单细胞蛋白的发酵提供优良菌株。【方法】从土壤、奶制品、水果采集样品分离酵母菌,根据形态学和分子生物学鉴定菌株,然后以硫酸铵为唯一氮源培养基,测定菌落大小、菌体干重、蛋白质含量,复筛氨氮利用率高的酵母菌,并对复筛菌株氨同化相关酶活性进行测定。【结果】经过形态学、分子生物学鉴定和氨氮利用能力评价,获得3株高氨氮利用的酵母菌,分别是胶红酵母(Rhodotorula mucilaginosa)、酿酒酵母(Saccharomyces cerevisiae)和戴尔有孢圆酵母(Torulaspora delbrueckii)。通过比较3株酵母菌的谷氨酸脱氢酶、谷氨酸合成酶、谷氨酰胺合成酶活性,酿酒酵母的3种酶活性最高,其次是胶红酵母。【结论】从奶酪和西瓜中分离的胶红酵母N5和酿酒酵母J1具有较强的氨氮利用能力以及酶活性,可为单细胞蛋白发酵提供优良菌株。     

Transposon mutations in the 5' end of glnD, the gene for a nitrogen regulatory sensor, that suppress the osmosensitive phenotype caused by otsBA lesions in Escherichia coli

GlnD of Escherichia coli is a bifunctional signal-transducing enzyme (102.4 kDa) which uridylylates the allosteric regulatory protein PII and deuridylylates PII-UMP in response to growth with nitrogen excess or limitation, respectively. GlnD catalyzes these reactions in response to high or low levels of cytoplasmic glutamine, respectively, and indirectly directs the expression of nitrogen-regulated genes, e.g., the glnK-amtB operon. We report that chromosomal mini-Tn10 insertions situated after nucleotide number 997 or 1075 of glnD partially suppressed the osmosensitive phenotype of DeltaotsBA or otsA::Tn10 mutations (defective osmoregulatory trehalose synthesis). Strains carrying these glnD::mini-Tn10 mutations either completely repressed the expression of trp::(glnKp-lacZ) or induced this reporter system to nearly 60% of the wild-type glnD level in response to nitrogen availability, an essentially normal response. This was in contrast to the much-studied glnD99::Tn10 mutation, which carries its insertion in the 3' end of the gene, causes a complete repression of glnKp-lacZ expression under all growth conditions, and also confers leaky glutamine auxotrophy. When expressed from the Pm promoter in plasmid constructs, the present glnD mutations produced proteins with an apparent mass of 39 or 42 kDa. These proteins were deduced to comprise 344 or 370 N-terminal residues, respectively, harboring the known nucleotidyltransferase domain of GlnD, plus a common C-terminal addition of 12 residues encoded by IS10. They lacked three other domains of GlnD. Apparently, the transferase domain by itself enabled the cells to catalyze the uridylylation reaction and direct nitrogen-regulated gene expression. Our data indicate that there exists a link between osmotic stress and the nitrogen response.     

Possible growth hormone regulation of rat liver glutamine synthetase activity.

Hypophysectomy results in a marked decrease in glutamine synthetase activity of rat liver homogenates. The enzyme is affected to a lesser extent in the kidneys and is not influenced in the brain. Bovine growth hormone treatment of hypophysectomized rats elevates the diminished glutamine synthetase activity in liver and kidneys but has no effect on the brain enzyme. Adrenalectomy also results in decreased liver glutamine synthetase activity although less than the decline seen with hypophysectomy. Cortisol treatment has no effect on glutamine synthetase activity in hypophysectomized animals. Our results suggest that growth hormone is involved in the regulation of liver glutamine synthetase activity. This regulation may be important in the utilization of α-amino nitrogen from glucogenic amino acids associated with growth hormone enhanced glucose production.     

Regulation of nitrate assimilation in the cyanobacterium Phormidium laminosum

The intracellular ratio of 2-oxoglutarate to glutamine has been analyzed under nutritional conditions leading to different activity levels of nitrate-assimilating enzymes in Phormidium laminosum (Agardh) Gom. This non-N₂-fixing cyanobacterium adapted to the available nitrogen source by modifying its nitrate reductase (NR; EC 1.7.7.2), nitrite reductase (NiR; EC 1.7.7.1) and glutamine synthetase (GS; EC 6.3.1.2) activities. The 2-oxoglutarate/glutamine ratio was similar in cells adapted to grow with nitrate or ammonium. However, metabolic conditions that increased this ratio [i.e., nitrogen starvation or l-methionine-d,l-sulfoximine (MSX) treatment] corresponded to high activity levels of NR, NiR, GS (except in MSX-treated cells) and glutamate synthase (GOGAT; EC 1.4.7.1). By contrast, metabolic conditions that diminished this ratio (i.e., addition of ammonium to nitrate-growing cells or addition of nitrate or ammonium to nitrogen-starved cells) resulted in low activity levels. The variation in the 2-oxoglutarate/glutamine ratio preceded the changes in enzyme activities. These results suggest that changes in the 2-oxoglutarate/glutamine ratio could be the signal that triggers the adaptation of P. laminosum cells to variations in the available nitrogen source, as occurs in enterobacteria.Abbreviations Chl chlorophyll - GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - GS glutamine synthetase (EC 6.3.1.2) - MSX l-methionine-d,l-sulfoximine - NiR nitrite reductase (EC 1.7.7.1) - NR nitrate reductase (EC 1.7.7.2) - TP total protein This work has been partially supported by grants from the Spanish Ministry of Education and Science (DGICYT PB88-0300 and PB92-0464) and the University of the Basque Country (042.310-EC203/94). M.I.T. was the recipient of a fellowship from the Basque Government.     

Sensitivity and Robustness in Covalent Modification Cycles with a Bifunctional Converter Enzyme

Regulation by covalent modification is a common mechanism to transmit signals in biological systems. The modifying reactions are catalyzed either by two distinct converter enzymes or by a single bifunctional enzyme (which may employ either one or two catalytic sites for its opposing activities). The reason for this diversification is unclear, but contemporary theoretical models predict that systems with distinct converter enzymes can exhibit enhanced sensitivity to input signals whereas bifunctional enzymes with two catalytic sites are believed to generate robustness against variations in system’s components. However, experiments indicate that bifunctional enzymes can also exhibit enhanced sensitivity due to the zero-order effect, raising the question whether both phenomena could be understood within a common mechanistic model. Here, I argue that this is, indeed, the case. Specifically, I show that bifunctional enzymes with two catalytic sites can exhibit both ultrasensitivity and concentration robustness, depending on the kinetic operating regime of the enzyme’s opposing activities. The model predictions are discussed in the context of experimental observations of ultrasensitivity and concentration robustness in the uridylylation cycle of the PII protein, and in the phosphorylation cycle of the isocitrate dehydrogenase, respectively.     

Sensitivity and Robustness in Covalent Modification Cycles with a Bifunctional Converter Enzyme

Regulation by covalent modification is a common mechanism to transmit signals in biological systems. The modifying reactions are catalyzed either by two distinct converter enzymes or by a single bifunctional enzyme (which may employ either one or two catalytic sites for its opposing activities). The reason for this diversification is unclear, but contemporary theoretical models predict that systems with distinct converter enzymes can exhibit enhanced sensitivity to input signals whereas bifunctional enzymes with two catalytic sites are believed to generate robustness against variations in system’s components. However, experiments indicate that bifunctional enzymes can also exhibit enhanced sensitivity due to the zero-order effect, raising the question whether both phenomena could be understood within a common mechanistic model. Here, I argue that this is, indeed, the case. Specifically, I show that bifunctional enzymes with two catalytic sites can exhibit both ultrasensitivity and concentration robustness, depending on the kinetic operating regime of the enzyme’s opposing activities. The model predictions are discussed in the context of experimental observations of ultrasensitivity and concentration robustness in the uridylylation cycle of the PII protein, and in the phosphorylation cycle of the isocitrate dehydrogenase, respectively.     

Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment.