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2.1.3.2: aspartate carbamoyltransferase

This is an abbreviated version!
For detailed information about aspartate carbamoyltransferase, go to the full flat file.

Word Map on EC 2.1.3.2

Reaction

Carbamoyl phosphate
+
L-aspartate
=
phosphate
 +
N-carbamoyl-L-aspartate

Synonyms

(S)-2-methyl-3-oxopropanoyl-CoA:pyruvate carboxyltransferase, ACT, aspartate carbamoyltransferase, aspartate carbamyltransferase, aspartate trans carbamoylase, aspartate transcarbamoylase, aspartate transcarbamylase, aspartic acid transcarbamoylase, aspartic carbamyltransferase, aspartic transcarbamylase, ATC, ATC domain of CAD, ATCase, CAD, carbamoylaspartotranskinase, carbamoyltransferase, aspartate, carbamylaspartotranskinase, L-aspartate transcarbamoylase, L-aspartate transcarbamylase, MJ1581, PYRB

ECTree

     2 Transferases
         2.1 Transferring one-carbon groups
             2.1.3 Carboxy- and carbamoyltransferases
                2.1.3.2 aspartate carbamoyltransferase

Crystallization

Crystallization on EC 2.1.3.2 - aspartate carbamoyltransferase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
complex of aspartate transcarbamoylase and dihydroorotase
noncovalent hexamer of dihydroorotase and ATCase, to 2.3 A resolution. The structure has citrate, bound to the active sites of both enzymes.Six DHO and six ATC chains form a hollow dodecamer, in which the 12 active sites face an internal reaction chamber that is approximately 60 A in diameter and connected to the cytosol by narrow tunnels. The entrances and the interior of the chamber are both electropositive, which suggests that the architecture of this nanoreactor modifies the kinetics of the bisynthase, not only by steric channeling but also by preferential escape of the product, dihydroorotase
50 mM Tis-HCl, pH 8.1, 70% ammonium sulfate
-
purified recombinant enzyme alone or complex with substrate carbamoyl phosphate or inhibitor N-phosphonacetyl-L-aspartate, hanging drop vapor diffusion method, 12 mg/ml protein is mixed with an equal volume of crystallization buffer containing 1.7 M (NH4)2SO4, 0.1 M Tris-HCl, pH 8.5, and 2.0% PEG 200, and equilibratopn over a reservoir of 0.5 ml of crystallization buffer at 20°C, 1 week, for substrate bound enzyme the crystals are soaked in mother liquor containing the ligand at 13.3 mM, for inhibitor bound form, the enzyme is crystallized as described using crystallization buffer containing 0.1 M potassium bromide, 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid, pH 10.0, and 18% PEG 8000, 20°C, 1 week, X-ray diffraction structure determination and analysis at 2.1-2.6 A resolution
approx. 30% ammonium sulfate, crystals appear after about 2 weeks at 4°C
-
ATCase locked in the R quaternary structure by specific introduction of disulfide bonds bound to the final product molecule phosphate, 10 mg/ml protein solution is dialyzed against a solution of 100 mM potassium dihydrogen phosphate and 3 mM sodium azide, pH 5.9, 1 week, X-ray diffraction structure determination and analysis at 2.85 A resolution, molecular replacement
-
bound to N-phosphonacetyl-L-aspartate, citrate or phosphate, crystalline R-state P212121
-
cocrystallization of catalytic subunit with N-(phosphonoacetyl)-L-aspartate by vapor diffusion, crystals grow from 0.005 ml of 100 mM Tris-HCl, pH 6.8, 20 mM calcium acetate, 5.8% polyethylene glycol 8000 and 0.005 ml of catalytic subunit in 10 mM Tris-HCl, pH 7.5, 1 mM 2-mercaptoethanol and 2 mM N-(phosphonoacetyl)-L-aspartate
hanging drop vapor diffusion method, using 0.1 M MES, pH 6.0, 10% (v/v) glycerol, and 10% (w/v) PEG 8000
hanging drop vapor diffusion method, using 0.2 m NH4Ac, 0.1 m Tris pH 8.5, 20% (w/v) PEG3350, and 10% glycerol for all enzyme mutants, and 0.1 m HEPES pH 7.0, 30% (w/v) Jeffamine M-600 pH 7.0, and 10% (v/v) glycerol for all enzyme holo mutants
hanging drop vapor diffusion, X-ray structure of unliganded ATCase at pH 8.5
-
microdialysis method, structure of D236A ATCase in the presence of phosphonoactamide and Asp
-
mutant E50A, in presence of phosphonoacetamide and malonate to trap the enzyme in T-like and R-like structures
-
mutant K244N, loss of numerous local T-state stabilizing interactions
-
purified recombinant ATCase in complex with UTP, CTP, or dCTP, dialysis of 20 mg/ml protein against 40 mM sodium citrate, 1 mM 2-mercaptoethanol, 0.2 mM EDTA, and 1.0 mM CTP, pH 5.7, at 20°C, 1 week, transfer of dialysis buttons to 2 mL of crystallization buffer with 5 mM UTP and 5 mM MgCl2 and equilibration for 12 h, and to crystallization buffer containing 5 mM UTP and 5 mM MgCl2 for 12 h, respectively, cryoprotection in 20% 2-methyl-2,4-pentanediol in UTP-Mg2+ crystallization buffer, X-ray diffraction structure determination and analysis at 1.9-2.1 A resolution
purified recombinant enzyme, 20 mg/ml protein is mixed with 40 mM sodium citrate, 1 mM 2-mercaptoethanol, 0.2 mM EDTA, and 1.0 mM CTP, pH 5.7, at 20 °C, 1 week, X-ray diffraction structure determination and analysis at 2.1 A resolution
purified recombinant mutant enzyme K164E/E239K, mixing of 10 mg/ml enzyme in 50 mM Tris-acetate, pH 8.3, with 0.002 ml of crystallization buffer containing 16% w/v PEG 4000, 0.04 M Na2MoO4-2H2O, 0.04 M N-cyclohexyl-3-aminopropanesulfonic acid, and 30 mM Tris-acetate, pH 8.75, and equilibration over a reservoir of crystallization buffer of 1.0 ml, 20°C, 2 weeks, X-ray diffraction structure determination and analysis
series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues, structure analysis, detailed overview. The structure of the enzyme in the presence of citrate, an analogue of N-carbamoyl-L-aspartate plus the product phosphatewas determined after displacement of N-phosphonacetyl-L-aspartate from R-state crystals
-
use of Methyl-TROSY spectra for assignment for approximately 60% of the observed methyl groups in TROSY maps of ATCase by the divide and conquer method. The combination of all approaches leads to assignments for 86% of the methyl groups, providing a large number of probes of structure and dynamics. The derived assignments are used to interpret chemical shift changes of ATCase upon titration with the nucleotide ATP. Large shift changes in the N-terminal tails of the regulatory chain provide the first evidence for structural perturbations in a region playing a critical role on the effect of nucleotide binding on distal catalytic sites of the allosteric enzyme
-
X-ray crystal structure of the N-phosphonacetyl-L-asparagine complexed with ATCase. Analysis of the crystal structure of the enzyme in the presence of N-phosphonacetyl-L-asparagine reveals that the binding of N-phosphonacetyl-L-asparagine is similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme
-
X-ray structure at 5.5 A resolution
-
X-ray structure of the holoenzyme in the presence of the substrate analog N-phosphonoacetyl-L-aspartate, carbamoylphosphate and succinate
-
X-ray structures of wild-type Escherichia coli aspartate transcarbamoylase holoenzyme and catalytic subunit crystallized with different ligands, overview
-
free enzyme and bound to carbamoyl phosphate or N-phosphonacetyl-L-aspartate
purified detagged isolated recombinant huATCase, free or in complex with inhibitor N-phosphonoacetyl-L-aspartate, 1.5 mg/ml protein, 0.1 M Tris pH 8.5, 6% PEG 8000, and 9% ethylene glycol is mixed with 0.1 M CAPS pH 10, 150 mM NaCl or 1.5 mg ml1 and 10 mM ZnCl2, 15% PEG 6000, 50 mM sodium acetate pH 4.8, and 0.5 mM inhibitor, X-ray diffraction structure determination and analysis at 2.1 A resolution
catalytic chain in the presence of the regulatory chain in the hexagonal space group P6322, with one monomer per asymmetric unit, sitting drop vapor diffusion method, mixing of 0.0013 protein-ligand solution, containing 11 mg/ml protein in 50 mM Tris, pH 8.3, 150 mM NaCl, 2 mM BME, 0.05 mM zinc acetate, with 0.001 ml of reservoir solution containing 2.0 M ammonium sulfate, 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M sodium citrate tribasic dihydrate, pH 5.6, or 2.0 M ammonium sulfate, 0.2 M potassium sodium tartrate tetrahydrate, 0.1 M Tris-HCl, pH 7.5, 22°C, X-ray diffraction structure determination and analysis at 2.50 A resolution, molecular replacement
crystals are grown at 295 K by the sitting-drop method from reservoirs containing 2.0 M ammonium sulfate and 5% 2-propanol. The catalytic trimer is crystallized in space group R32, with unit-cell parameters a = b = 265.3, c = 195.5 A and two trimers in the asymmetric unit. Its structure is determined using molecular replacement and Patterson methods
crystals of the catalytic subunit in an orthorhombic crystal form contain four crystallographically independent trimers which associate in pairs to form stable staggered complexes. Each subunit has a sulfate in the central channel. The catalytic subunits in these complexes show flexibility, with the elbow angles of the monomers differing by up to 7.4° between crystal forms. There is also flexibility in the relative orientation of the trimers around their threefold axis in the complexes, with a difference of 4° between crystal forms
sitting drop method, crystal structure of the catalytic trimer
3 catalytic and 3 regulatory subunits per asymmetric unit
-
the crystal structure of the unliganded Moritella profunda ATCase shows resemblance to a more extreme T state reported previously for an Escherichia coli ATCase mutant
-
full-length enzyme, sitting drop vapor diffusion method, using 0.2 M potassium citrate tribasic monohydrate, 20% (w/v) PEG 3350. Enzyme in complex with 2,3-naphthalenediol, hanging drop vapor diffusion method, 0.1 M bis-Tris propane pH 7.5, 0.2 M Na2SO4, 15% (w/v) PEG 3350
truncated enzyme, hanging drop vapor diffusion method, using 0.2 M sodium sulfate, 5 mM MgSO4, 15% (w/v) PEG 3350 in 0.1 M bis-Tris propane pH 7.5
hanging drop method, equal volumes of 1 mg/ml enzyme in 100 mM Tris-HCl, pH 8.5 is mixed with 100 mM citrate, pH 5, and 10% 6K polyethylene glycol
-
hanging-drop vapour-diffusion, 0.002 ml of enzyme solution, 7 mg/ml, in 20 mM Tris-HCl, pH 8.2, 2 mM 2-mercaptoethanol, 300 mM NaCl, is mixed with 0.002 ml reservoir solution consisting of 1.4 M citrate, pH 6.8, X-ray structure to 1.8 A resolution
aspartate carbamoyltransferase in complex with its allosteric activator CTP
-
hanging drop vapour-diffusion technique at 20°C. Tertiary and quaternary structure of the T state ATCase of the enzyme determined by X-ray crystallography to 2.6 A resolution
-
in ligand free form and in complex with carbamoyl phosphate to 2.8 A and to 1.6 A resolution, respectively. Presence of two homotrimers in the asymmetric unit
-