Inhibition of Escherichia coli CTP synthase by glutamate γ-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis

2001 ◽  
Vol 356 (1) ◽  
pp. 223-232 ◽  
Author(s):  
Stephen L. BEARNE ◽  
Omid HEKMAT ◽  
Jennifer E. MacDONNELL
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Specific Activity ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Open Chain ◽  
Tetrahedral Intermediate ◽  
Allosteric Effector ◽  
Ctp Synthase

Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate γ-semialdehyde. Glutamate γ-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate γ-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (Kis 0.16±0.03mM; Kii 0.4±0.1mM) and a competitive inhibitor with respect to ammonia (Ki 0.39±0.06mM) in the presence of GTP at pH8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate γ-semialdehyde. Although glutamate γ-semialdehyde exists in solution primarily in its cyclic form, Δ1-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Δ1-pyrroline-5-carboxylate (l-proline, l-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate γ-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

Preparation of Isotope-labeled Dihydroneopterin 3'-Triphosphate with High Specific Activity

Pteridines ◽  
1990 ◽  
Vol 2 (3) ◽  
pp. 169-174 ◽  
Author(s):  
Gerd Katzenmeier ◽  
Bruno Schwarzkopf ◽  
Quang Le Van ◽  
Cornelia Schmid ◽  
Adelbert Bacher
Keyword(s):  
Escherichia Coli ◽  
Adenylate Kinase ◽  
Specific Activity ◽  
High Specific Activity ◽  
Starting Material ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Guanylate Kinase ◽  
Phosphoribosyl Transferase ◽  
Phosphoribosylpyrophosphate Synthetase

SummaryIsotope-labeled dihydroneopterin 3'-triphosphate with 3H at positions C-1' and C-2', respectively, has been prepared from isotope-labeled glucose as starting material. Glucose was first converted enzymatically to ribose 5-phosphate. GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase. It was subsequently phosphorylated to GTP in two steps using adenylate kinase and guanylate kinase. Dihydroneopterin triphosphate was prepared from GTP by the action of recombinant GTP-cyclohydrolase I from Escherichia coli. The method allows the incorporation of 3H and 14C isotope labels into any desired position of dihydroneopterin triphosphate. Rapid purfication procedures for phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase as well as HPLC assays for their determinations are described.

scholarly journals Aspartate-107 and leucine-109 facilitate efficient coupling of glutamine hydrolysis to CTP synthesis by Escherichia coli CTP synthase

2003 ◽  
Vol 369 (3) ◽  
pp. 497-507 ◽  
Author(s):  
Akshai IYENGAR ◽  
Stephen L. BEARNE
Keyword(s):  
Escherichia Coli ◽  
Normal Structure ◽  
Kinetic Properties ◽  
Wild Type ◽  
Nucleotide Binding Sites ◽  
Allosteric Effector ◽  
Close Proximity ◽  
Ctp Synthase ◽  
Dependent Activity ◽  
Glutaminase Activity

CTP synthase catalyses the ATP-dependent formation of CTP from UTP using either NH3 or l-glutamine as the nitrogen source. GTP is required as an allosteric effector to promote glutamine hydrolysis. In an attempt to identify nucleotide-binding sites, scanning alanine mutagenesis was conducted on a highly conserved region of amino acid sequence (residues 102—118) within the synthase domain of Escherichia coli CTP synthase. Mutant K102A CTP synthase exhibited wild-type activity with respect to NH3 and glutamine; however, the R105A, D107A, L109A and G110A enzymes exhibited wild-type NH3-dependent activity and affinity for glutamine, but impaired glutamine-dependent CTP formation. The E103A, R104A and H118A enzymes exhibited no glutamine-dependent activity and were only partially active with NH3. Although these observations were compatible with impaired activation by GTP, the apparent affinity of the D107A, L109A and G110A enzymes for GTP was reduced only 2—4-fold, suggesting that these residues do not play a significant role in GTP binding. In the presence of GTP, the kcat values for glutamine hydrolysis by the D107A and L109A enzymes were identical with that of wild-type CTP synthase. Overall, the kinetic properties of L109A CTP synthase were consistent with an uncoupling of glutamine hydrolysis from CTP formation that occurs because an NH3 tunnel has its normal structure altered or fails to form. L109F CTP synthase was prepared to block totally the putative NH3 tunnel; however, this enzyme's rate of glutamine-dependent CTP formation and glutaminase activity were both impaired. In addition, we observed that mutation of amino acids located between residues 102 and 118 in the synthase domain can affect the enzyme's glutaminase activity, suggesting that these residues interact with residues in the glutamine amide transfer domain because they are in close proximity or via a conformationally dependent signalling mechanism.

scholarly journals N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms

1999 ◽  
Vol 55 (7) ◽  
pp. 1350-1352 ◽  
Author(s):  
Fernando Gil ◽  
Santiago Ramón-Maiques ◽  
Alberto Marina ◽  
Ignacio Fita ◽  
Vicente Rubio
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
High Specific Activity ◽  
Unit Cell ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Space Group ◽  
Cell Parameters ◽  
E Coli ◽  
Carbamate Kinase

The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 µmol s−1 mg−1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 Å and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 Å) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 Å and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 Å), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.

scholarly journals Properties of a Thermostable Nitrate Reductase from the Hyperthermophilic Archaeon Pyrobaculum aerophilum

2001 ◽  
Vol 183 (19) ◽  
pp. 5491-5495 ◽  
Author(s):  
Sepideh Afshar ◽  
Eric Johnson ◽  
Simon de Vries ◽  
Imke Schröder
Keyword(s):  
Nitrate Reductase ◽  
Specific Activity ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Pyrobaculum Aerophilum ◽  
Benzyl Viologen ◽  
Hyperthermophilic Archaeon

ABSTRACT The nitrate reductase of the hyperthermophilic archaeonPyrobaculum aerophilum was purified 137-fold from the cytoplasmic membrane. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme complex consists of three subunits with apparent molecular weights of 130,000, 52,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), iron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofactors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific activity using reduced benzyl viologen as the electron donor (V max with nitrate, 1,162 s−1 (326 U/mg);V max with chlorate, 1,348 s−1 (378 U/mg) [assayed at 75°C]). The Km values for nitrate and chlorate were 58 and 140 μM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperature optimum for activity was >95°C. When incubated at 100°C, the purified nitrate reductase had a half-life of 1.5 h. This study constitutes the first description of a nitrate reductase from a hyperthermophilic archaeon.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

scholarly journals High-Level Extracellular Expression of a New β-N-Acetylglucosaminidase in Escherichia coli for Producing GlcNAc

2021 ◽  
Vol 12 ◽  
Author(s):  
Congna Li ◽  
Shun Jiang ◽  
Chao Du ◽  
Zhenghui Lu ◽  
Nisha He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Chemical Process ◽  
Specific Activity ◽  
High Specific Activity ◽  
Culture Conditions ◽  
Extracellular Expression ◽  
Secreted Expression ◽  
Encoding Gene ◽  
High Level ◽  
The Cost

N-acetyl-β-D glucosamine (GlcNAc) is wildly used in cosmetics, nutraceuticals and pharmaceuticals. The traditional chemical process for GlcNAc production from chitin causes serious acidic pollution. Therefore, the enzymatic hydrolysis becomes a great promising and alternative strategy to produce GlcNAc. β-N-acetylglucosaminidase (NAGase) can hydrolyze chitin to produce GlcNAc. Here, a GH3 family NAGase encoding gene BlNagZ from Bacillus licheniformis was expressed extracellularly in Escherichia coli guided by signal peptide PelB. The recombinant BlNagZ presented the best activity at 60°C and pH 5.5 with a high specific activity of 13.05 U/mg. The BlNagZ activity in the fermentation supernatant can reach 13.62 U/mL after optimizing the culture conditions, which is 4.25 times higher than optimization before. Finally, combining BlNagZ with chitinase ChiA we identified before, chitin conversion efficiency to GlcNAc can reach 89.2% within 3.5 h. In all, this study provided not only a high active NAGase, and a secreted expression strategy to reduce the cost of production, which is conducive to the industrial application.

Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity

Biochemistry ◽  
1992 ◽  
Vol 31 (30) ◽  
pp. 6917-6924 ◽  
Author(s):  
Kimberly Carter Minghetti ◽  
Visala Chepuri Goswitz ◽  
N. Elise Gabriel ◽  
John J. Hill ◽  
Carlos A. Barassi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Specific Activity ◽  
High Specific Activity ◽  
Terminal Oxidase ◽  
Cytochrome O

scholarly journals Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits

1997 ◽  
Vol 327 (3) ◽  
pp. 891-898 ◽  
Author(s):  
M. Craig HILL ◽  
Siew Siew PANG ◽  
G. Ronald DUGGLEBY
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Large Subunit ◽  
Plasmid Vector ◽  
Small Subunit ◽  
Enzymic Activity ◽  
Single Step ◽  
Acetohydroxyacid Synthase ◽  
Kinetic Properties ◽  
Fusion Enzyme

The first step in the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (EC 4.1.3.18). The reaction involves the decarboxylation of pyruvate followed by condensation with either a second molecule of pyruvate or with 2-oxobutyrate. The enzyme requires as cofactors thiamine diphosphate, a divalent metal ion and, usually, FAD. In most bacteria the enzyme is a heterotetramer of two large and two small subunits. Escherichia coli contains three active isoenzymes and the present study concerns isoenzyme II, whose large and small subunits are encoded by the ilvG and ilvM genes respectively. Cloning these genes into a plasmid vector and overexpression in E. coli allowed a two-step purification procedure for the native enzyme to be developed. The level of expression is considerably higher from a vector that introduces a 50 residue N-terminal fusion containing an oligohistidine sequence on the large subunit. Purification to homogeneity was achieved in a single step by immobilized-metal-affinity chromatography. The kinetic properties of the native and fusion enzyme are indistinguishable with respect to the substrate pyruvate and the inhibitor chlorsulfuron. The individual subunits were expressed as oligohistidine-tagged fusion proteins and each was purified in a single step. Neither subunit alone has significant enzymic activity but, on mixing, the enzyme is reconstituted. The kinetic properties of the reconstituted enzyme are very similar to those of the fusion enzyme. It is proposed that the reconstitution pathway involves successive, and highly co-operative, binding of two small subunit monomers to a large subunit dimer. None of the cofactors is needed for subunit association although they are necessary for the restoration of enzymic activity.

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