Glutamine Synthetase Cascade Research Articles

Modeling the role of covalent enzyme modification inEscherichia colinitrogen metabolism

In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times.

Modeling the role of covalent enzyme modification in Escherichia coli nitrogen metabolism

In the bacterium Escherichia coli, the enzyme glutamine synthetase (GS) converts ammonium into the amino acid glutamine. GS is principally active when the cell is experiencing nitrogen limitation, and its activity is regulated by a bicyclic covalent modification cascade. The advantages of this bicyclic-cascade architecture are poorly understood. We analyze a simple model of the GS cascade in comparison to other regulatory schemes and conclude that the bicyclic cascade is suboptimal for maintaining metabolic homeostasis of the free glutamine pool. Instead, we argue that the lag inherent in the covalent modification of GS slows the response to an ammonium shock and thereby allows GS to transiently detoxify the cell, while maintaining homeostasis over longer times.

The pivotal regulator GlnB of Escherichia coli is engaged in subtle and context‐dependent control

This study tests the purported signal amplification capability of the glutamine synthetase (GS) regulatory cascade in Escherichia coli. Intracellular concentrations of the pivotal regulatory protein GlnB were modulated by varying expression of its gene (glnB). Neither glnB expression nor P(II)* (i.e. the sum of the concentration of the P(II)-like proteins GlnB and GlnK) had control over the steady-state adenylylation level of GS when cells were grown in the presence of ammonia, in which glnK is not activated. Following the removal of ammonia, the response coefficient of the transient deadenylylation rate of GS-AMP was again zero with respect to both glnB expression and P(II)* concentration. This was at wild-type P(II)* levels. A 20% decrease in the P(II)* level resulted in the response coefficients increasing to 1, which was quite significant yet far from expected for zero-order ultrasensitivity. The transient deadenylylation rate of GS-AMP after brief incubation with ammonia was also measured in cells grown in the absence of ammonia. Here, GlnK was present and both glnB expression and P(II)* lacked control throughout. Because at wild-type levels of P(II)*, the molar ratio of P(II)*-trimer/adenylyltransferase-monomer was only slightly above 1, it is suggested that the absence of control by P(II)* is caused by saturation of adenylyltransferase by P(II)*. The difference in the control of deadenylylation by P(II)* under the two different growth conditions indicates that control of signal transduction is adjusted to the growth conditions of the cell. Adjustment of regulation rather than ultrasensitivity may be the function of signal transduction chains such as the GS cascade. We discuss how the subtle interplay between GlnB, its homologue GlnK and the adenylyltransferase may be responsible for the 'redundant', but quantitative, phenotype of GlnB.

Macromolecular intelligence in microorganisms.

Biochemistry and molecular biology have been focusing on the structural, catalytic, and regulatory properties of individual macromolecules from the perspective of clarifying the mechanisms of metabolism and gene expression. Complete genomes of 'primitive' living organisms seem to be substantially larger than necessary for metabolism and gene expression alone. This is in line with the findings of silent phenotypes for supposedly important genes, apparent redundancy of functions, and variegated networks of signal transduction and transcription factors. Here we propose that evolutionary optimization has been much more intensive than to lead to the bare minima necessary for autonomous life. Much more complex organisms prevail. Much of this complexity arises in the nonlinear interactions between cellular macromolecules and in subtle differences between paralogs (isoenzymes). The complexity can only be understood when analyzed quantitatively, for which quantitative experimentation is needed in living systems that are as simple and manipulatable as possible, yet complex in the above sense. We illustrate this for the glutamine synthetase cascade in Escherichia coli. By reviewing recent molecular findings, we show that this cascade is much more complex than necessary for simple regulation of ammonia assimilation. Simulations suggest that the function of this complexity may lie in quasi-intelligent behavior, including conditioning and learning.

An additional PIIinEscherichia coli: a new regulatory protein in the glutamine synthetase cascade

The PII protein in the glutamine synthetase cascade transduces the nitrogen signal, as sensed by uridylyltransferase, both to the NRII/NRI two-component system and to adenylyltransferase, to regulate the activity of glutamine synthetase. Here we describe the amplification of a chromosomal DNA fragment from Escherichia coli which contains the sequence of a PII homologue. The derived amino acid sequence of this DNA fragment is 67% identical to E. coli PII. It contains the conserved tyrosine residue which is known to be the site of uridylylation in PII. E. coli is the first organism in which two different PII proteins have been detected.

An additional P II in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade

The P II protein in the glutamine synthetase cascade transduces the nitrogen signal, as sensed by uridylyltransferase, both to the NRII/NRI two-component system and to adenylyltransferase, to regulate the activity of glutamine synthetase. Here we describe the amplification of a chromosomal DNA fragment from Escherichia coli which contains the sequence of a P II homologue. The derived amino acid sequence of this DNA fragment is 67% identical to E. coli P II. It contains the conserved tyrosine residue which is known to be the site of uridylylation in P II. E. coli is the first organism in which two different P II proteins have been detected.

In Situ Regulation Studies of Escherichia coli Glutamine Synthetase

The involvement of glutamine in the synthesis of purine and pyrimidine nucleotides, glucosamine 6-phosphate, and p -aminobenzoic acid, and in the formation of all the amino acids makes this metabolite a key intermediate in the synthesis of important end products such as proteins, nucleic acids, and complex polysaccharides. The enzyme of Escherichia coli and other gram-negative bacteria has been widely studied and appears to possess an extraordinary capability to integrate quite different metabolic signals and react appropriately. The E. coli glutamine synthetase (GS) is regulated at least by four different regulatory mechanisms, including repression of its synthesis, cumulative feedback inhibition by several end products of glutamine metabolism, modulation by the divalent metal ions, and covalent modification. With the aim of verifying in vivo the exceptional capability of GS to answer with an appropriate’ catalytic potency and regulatory susceptibility to different microenvironmental conditions, a method has been devised to study the GS cascade system in situ . E.coli can be made permeable to low molecular weight compounds while retaining GS and its cascade components by treating the cells with the nonionic detergent.

Rat liver 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase: a review of relationships between the two activities of the enzyme.

Both the synthesis and the degradation of Fru-2,6-P2 are catalyzed by a single enzyme protein; ie, the enzyme is bifunctional. This protein, which we have designated 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase is an important enzyme in the regulation of hepatic carbohydrate metabolism since its activity determines the steady-state concentration of fructose 2,6-P2, an activator of 6-phosphofructo 1-kinase and an inhibitor of fructose 1,6-bisphosphatase. Regulation of the bifunctional enzyme in intact cells is a complex function of both covalent modification via phosphorylation/dephosphorylation and the influence of substrates and low molecular weight effectors. Recent evidence suggests that both reactions may proceed by two-step transfer mechanisms with different phosphoenzyme intermediates. The enzyme catalyzes exchange reactions between ADP and ATP and between fructose 6-P and fructose 2,6-P2. A labeled phosphoenzyme is formed rapidly during incubation with [2-32P]Fru-2,6-P2. The labeled residue has been identified as 3-phosphohistidine. However, it was not possible to demonstrate significant labeling of the enzyme directly from [gamma-32P]ATP. These results can be most readily explained in terms of two catalytic sites, a kinase site whose phosphorylation by ATP is negligible (or whose E-P is labile) and a fructose 2,6-bisphosphatase site which is readily phosphorylated by fructose 2,6-P2. Additional evidence in support of two active sites include: limited proteolysis with thermolysin results in loss of 6-phosphofructo 2-kinase activity and activation of fructose 2,6-bisphosphatase, mixed function oxidation results in inactivation of the 6-phosphofructo 2-kinase but no affect on the fructose 2,6-bisphosphatase, N-ethylmaleimide treatment also inactivates the kinase but does not affect the bisphosphatase, and p-chloromercuribenzoate immediately inactivates the fructose 2,6-bisphosphatase but not the 6-phosphofructo 2-kinase. Our findings indicate that the bifunctional enzyme is a rather complicated enzyme; a dimer, probably with two catalytic sites reacting with sugar phosphate, and with an unknown number of regulatory sites for most of its substrates and products. Three enzymes from Escherichia coli, isocitric dehydrogenase kinase/phosphatase, glutamine-synthetase adenylyltransferase, and the uridylyltransferase for the regulatory protein PII in the glutamine synthetase cascade system also catalyze opposing reactions probably at two discrete sites. All four enzymes are important in the regulation of metabolism and may represent a distinct class of regulatory enzymes.

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 m m 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.

Glutamine synthetase cascade: Enrichment of uridylyltransferase in Escherichia coli carrying hybrid ColE1 plasmids

We have used hybrid ColE1 plasmids that carry small selected segments of the Escherichia coli chromosome ( L. Clarke and J. Carbon, 1976 Cell 9, 91–99 ) to obtain increased levels of two of the components involved in the covalent modification of glutamine synthetase. Screening of the plasmid collection for those strains capable of correcting the glutamine requirement in glnD mutants of E. coli which lack uridylyltransferase activity indicated that two of these plasmid-containing strains, JA200/pLC 6–32 and JA200/pLC 38–39, overproduce Uridylyltransferase by 14- to 25-fold. In both of these strains the increase in uridylyltransferase levels was correlated with a parallel increase in uridylyl-removing enzyme levels. This is in agreement with evidence which suggests that both of these activities may be encoded by the same or closely neighboring genes. Two plasmids (pLC 28–36 and pLC 41-35), that complement strains with mutations in the glnA gene, the structural gene for glutamine synthetase, are also capable of complementing the defect in the glnD mutants. However, as shown here, this occurs by a mechanism other than overproduction of uridylyltransferase.

New methods for the colorimetric assay of P IID 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 Mn 2+). 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.