Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene

A procedure was developed to purify large quantities of PII protein from an Escherichia coli strain which contains a multicopy plasmid harboring the structural gene of PII (the glnB gene). Ultraviolet spectra of uridylylated and unuridylylated PII were obtained using the purified PII and empirical formulas to calculate the concentration of protein and the average number of uridylylated subunits per molecule were derived. A continuous fluorometric assay for the measurement of uridylylated PII (PIID) and adenylyltransferase (ATase) was also established. Rate measurements at various concentrations of PIID and at a fixed concentration of ATase showed that a tetrameric PIID molecule interacts with only one ATase molecule at a time. The complete nucleotide sequence of the glnB gene was determined and parts of the deduced amino acid sequence were confirmed by the results of amino acid sequence analysis of peptides. The PII subunit consists of 103 amino acids (Mr = 11,580). Two tyrosines reside at positions 46 and 51, where Tyr51 is the site of uridylylation. Nucleotide sequence analysis of the upstream region showed no obvious sites for the binding of RNA polymerase, indicating that the glnB gene is a part of an as yet unidentified operon.     

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.     

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.     

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.     

Crystals of glutamine synthetase from Escherichia coli.

Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are $\text{a = 140 ± 2}\text{A}\text{̊}$, and $\text{c = 148 ± 2}\text{A}\text{̊}$. The Laue symmetry group is $\text{3}\text{̄}$m P3₁.     

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.     

Some properties of Escherichia coli glutamine synthetase after limited proteolysis by subtilisin.

Escherichia coli glutamine synthetase is inactivated by subtilisin. Protection against inactivation is afforded by glutamine and ammonium ions. One large fragment (Mr = 35,000) is identified by sodium dodecyl sulfate-gel electrophoresis and carries adenylylation site. Smaller quantities of two other fragments (Mr = 17,000 and 15,000, respectively) are als observed oo observed on the gel. tthe nicked protein remains dodecameric, as evidenced by electrophoresis and centrifugation. It has retained the binding properties toward ADP and Ci-bacron blue and undergoes conformation changes upon binding, as does the intact protein. It is recognized by the antiserum raised against the native enzyme. The nicked protein also remains an excellent substrate of E. coli adenylyltransferase.     

Purification and properties of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli

1. Methionyl-t-RNA synthetase (where t-RNA denotes ;soluble' or transfer RNA) has been purified to apparent homogeneity from a ribonuclease I-free strain of Escherichia coli. Polyacrylamide-gel electrophoresis of the final product revealed a single band. The purified enzyme catalyses the exchange of 450mumoles of pyrophosphate into ATP/mg. in 15min. at 37 degrees . 2. Methionyl-t-RNA synthetase is specific for the l-isomer of methionine, but appears to catalyse the methionylation of two distinct species of t-RNA, both of which are specific for methionine, but only one of which may be subsequently formylated. 3. The Michaelis constant for l-methionine is 2x10(-4)m in the ATP-PP(i) exchange assay and 2x10(-5)m for the acylation of t-RNA. 4. Gel filtration of both crude and highly purified preparations of methionyl-t-RNA synthetase on Sephadex G-200 indicates that the active species of enzyme has a molecular weight of about 190000. The amino acid composition of the enzyme is similar to those reported for the isoleucine and tyrosine enzymes from E. coli.     

Purification of a protease from Escherichia coli with specificity for oxidized glutamine synthetase

A soluble Escherichia coli protease has been identified and purified to homogeneity. The protease cleaves glutamine synthetase which has been modified by mixed function oxidation; native glutamine synthetase is not a substrate. Using [14C]glutamine synthetase as a substrate (prepared by growing E. coli on 14C-labeled amino acids), protease activity was assayed by determining the release of trichloroacetic acid-soluble material. The pure protease cleaves glutamine synthetase near the carboxyl terminus yielding 4,500 and 47,000 Mr products. The characteristics of this enzyme distinguish it from proteases previously purified from E. coli. These characteristics include a molecular weight of 75,000, alkaline pH optimum, lack of inhibition by serine protease inhibitors, and the ability to degrade insulin and casein. Oxidation of glutamine synthetase and other enzymes can be catalyzed by a variety of mixed function oxidase systems from bacterial and mammalian sources. Mixed function oxidation may be a "signal" or "marker" which consigns a protein for proteolytic degradation. Susceptibility to oxidation is subject to metabolic regulation, thereby providing control of proteolytic turnover. Isolation of a protease specific for modified glutamine synthetase provides the enzymatic basis for the specificity of this scheme.     

Regulation of enzyme formation in Klebsiella aerogenes by episomal glutamine synthetase of Escherichia coli.

We studied the physiology of cells of Klebsiella aerogenes containing the structural gene for glutamine synthetase (glnA) of Escherichia coli on an episome. The E. coli glutamine synthetase functioned in cells of K. aerogenes in a manner similar to that of the K. aerogenes enzyme: it allowed the level of histidase to increase and that of glutamate dehydrogenase to decrease during nitrogen-limited growth. The phenotype of mutations in the glnA site was restored to normal by the introduction of the episomal glnA+ gene. These results are consistent with the hypothesis that glutamine synthetase regulates the function of its own structural gene.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.