EC Number | Cloned (Comment) | Organism |
---|---|---|
2.4.2.18 | wild-type and mutants in pQE40 for expression with N-terminal hexa-His tag in Escherichia coli W3110 trpEA2 (pDM) | Saccharolobus solfataricus |
EC Number | Crystallization (Comment) | Organism |
---|---|---|
2.4.2.18 | mutant M47D, structurally very similar to wild-type (rms deviation of 0.7 A for most of equivalent C(alpha) atoms) but reduced buried surface area per subunit compared to wild-type homodimer, Aps47 protonated at pH 6, crystals of space group P2 with four molecules (two homodimers) per asymmetric unit and A2 pseudo-symmetry, unit cell parameters a=91.6 A, b=65.9 A, c=115.7 A, beta=107.4°, 45% (v/v) solvent content, hanging drop method: 1 microlitre protein solution (5 mg/ml) + 1 microlitre reservior solution (50 mM MES pH 6.0, 18% (v/v) PEG, 5% (v/v) glycerol), room temperature, 72 h | Saccharolobus solfataricus |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.4.2.18 | I36E | weakened intersubunit interaction and increased protein solubility by introduction of negative side chain, monomer-dimer equilibrium (dissociation constant, KD: 0.8 +/-0.6 microM), concentration-dependent kinetic stability during heat inactivation (80°C) with half-lives from 3 to 40 min, no suitable crystal formed | Saccharolobus solfataricus |
2.4.2.18 | M47D | weakened intersubunit interaction and increased protein solubility by introduction of negative side chain, monomer-dimer equilibrium (dissociation constant, KD: 17 +/-10 microM), concentration-dependent kinetic stability during heat inactivation (80°C) with half-lives from 4 to 15 min, no structural perturbation | Saccharolobus solfataricus |
2.4.2.18 | M47D/I36E | double mutant, monomeric, similar catalytic efficiencies as the wild-type for both substrates, first-order kinetics for time-dependent but not concentration-dependent heat inactivation at 80°C with half-live t1/2: 3 min, no suitable crystal formed | Saccharolobus solfataricus |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.4.2.18 | 0.000058 | - |
anthranilate | I36E/M47D double mutant, pH 7.2, 37°C, 0.01 microM subunit concentration | Saccharolobus solfataricus | |
2.4.2.18 | 0.000085 | - |
anthranilate | wild-type, pH 7.2, 37°C, 0.01 microM subunit concentration | Saccharolobus solfataricus | |
2.4.2.18 | 0.15 | - |
5-phospho-alpha-D-ribose 1-diphosphate | I36E/M47D double mutant, pH 7.2, 37°C, 0.01 microM subunit concentration | Saccharolobus solfataricus | |
2.4.2.18 | 0.18 | - |
5-phospho-alpha-D-ribose 1-diphosphate | wild-type, pH 7.2, 37°C, 0.01 microM subunit concentration | Saccharolobus solfataricus |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
2.4.2.18 | Mg2+ | essential | Saccharolobus solfataricus |
EC Number | Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|---|
2.4.2.18 | 32400 | - |
double mutant M47D/I36E, analytical-gel filtration chromatography | Saccharolobus solfataricus |
2.4.2.18 | 34100 | - |
double mutant M47D/I36E, analytical ultracentrifugation (sedimentation equilibrium) | Saccharolobus solfataricus |
2.4.2.18 | 38800 | - |
calculated for monomeric wild-type and mutants | Saccharolobus solfataricus |
2.4.2.18 | 60100 | - |
mutant M47D, analytical ultracentrifugation (sedimentation equilibrium) | Saccharolobus solfataricus |
2.4.2.18 | 61700 | - |
mutant M47D, analytical gel filtration chromatography | Saccharolobus solfataricus |
2.4.2.18 | 63400 | - |
mutant I36E, analytical gel filtration chromatography | Saccharolobus solfataricus |
2.4.2.18 | 64500 | - |
wild-type, analytical gel filtration chromatography | Saccharolobus solfataricus |
2.4.2.18 | 66000 | - |
mutant I36E, analytical ultracentrifugation (sedimentation equilibrium) | Saccharolobus solfataricus |
2.4.2.18 | 72200 | - |
wild-type, analytical ultracentrifugation (sedimentation equilibrium) | Saccharolobus solfataricus |
2.4.2.18 | 77700 | - |
calculated for the dimeric wild-type and mutants | Saccharolobus solfataricus |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.2.18 | anthranilate + 5-phospho-alpha-D-ribose 1-diphosphate | Saccharolobus solfataricus | tryptophan biosynthesis, class III phosphoribosyltransfer | N-(5-phospho-D-ribosyl)-anthranilate + diphosphate | - |
ir |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.4.2.18 | Saccharolobus solfataricus | P50384 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.4.2.18 | from Escherichia coli extract by heat-precipitation of host proteins followed by metal chelate affinity chromatography, yield of 0.4-0.8 mg protein per 1 g wet cell mass, >95% purity, stored at -80°C | Saccharolobus solfataricus |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.2.18 | anthranilate + 5-phospho-alpha-D-ribose 1-diphosphate | tryptophan biosynthesis, class III phosphoribosyltransfer | Saccharolobus solfataricus | N-(5-phospho-D-ribosyl)-anthranilate + diphosphate | - |
ir | |
2.4.2.18 | anthranilate + 5-phospho-alpha-D-ribose 1-diphosphate | 37°C, pH 7.2, 0.05 mM Mg2+ | Saccharolobus solfataricus | N-(5-phospho-D-ribosyl)-anthranilate + diphosphate | - |
ir |
EC Number | Subunits | Comment | Organism |
---|---|---|---|
2.4.2.18 | dimer | wild-type and mutants I36E and M47D, analytical gel filtration chromatography and analytical ultracentrifugation (sedimentation equilibrium), dimerisation mediated by residues of mainly alpha helices 1, 3 and 8 of the small alpha-helical domains (one in each subunit) in head-to-head fashion | Saccharolobus solfataricus |
2.4.2.18 | monomer | double mutant M47D/I36E, analytical gel filtration chromatography and analytical ultracentrifugation (sedimentation equilibrium), Ile36 and Met47 situated at N-terminus and C-terminus of helix 3 of the small alpha-helical domain and involved in intimate intersubunit interactions | Saccharolobus solfataricus |
2.4.2.18 | monomer or dimer | equilibrium between minor, thermo-labile monomeric and major, thermo-stable dimeric state of single mutants M47D (dissociation constant, KD: 17 +/-10 microM) and I36E (KD: 0.8 +/-0.6 microM), analytical ultracentrifugation (sedimentation equilibrium) | Saccharolobus solfataricus |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.4.2.18 | AnPRT | - |
Saccharolobus solfataricus |
2.4.2.18 | anthranilate phosphoribosyltransferase | - |
Saccharolobus solfataricus |
2.4.2.18 | ssAnPRT | - |
Saccharolobus solfataricus |
2.4.2.18 | ssTrpD | - |
Saccharolobus solfataricus |
EC Number | Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|---|
2.4.2.18 | additional information | - |
concentration-dependent kinetic stability of single mutants I36E and M47D during heat inactivation at 80°C | Saccharolobus solfataricus |
2.4.2.18 | additional information | - |
dimerisation stabilizes against thermal denaturation | Saccharolobus solfataricus |
2.4.2.18 | 80 | - |
half-lives (t1/2) as measure of kinetic stability, t1/2(wild-type): 40 min, t1/2(double mutant M47D/I36E): 3 min, t1/2(mutant I36E, about 1 microM): 3 min, t1/2(mutant I36E, about 20 microM): 40 min, t1/2(mutant M47D, about 1 microM): 4 min, t1/2(mutant M47D, about 47 microM): 15 min, irreversible heat inactivation (pH 6.7) at different time points followed by chilling, centrifugation and estimation of residual activity | Saccharolobus solfataricus |
2.4.2.18 | 82 | - |
double mutant I36E/M47D, 12 microM, melting temperature at which half of the protein is unfolded deduced from DSC, pH 7.5 | Saccharolobus solfataricus |
2.4.2.18 | 83 | - |
mutant M47D, 12 microM, melting temperature at which half of the protein is unfolded deduced from DSC, pH 7.5 | Saccharolobus solfataricus |
2.4.2.18 | 90 | - |
mutant I36E, 12 microM, melting temperature at which half of the protein is unfolded deduced from DSC, pH 7.5 | Saccharolobus solfataricus |
2.4.2.18 | 92 | - |
wild-type, 12 microM, melting temperature at which half of the protein is unfolded deduced from differential scanning calorimetry (DSC), pH 7.5 | Saccharolobus solfataricus |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
2.4.2.18 | additional information | - |
5-phospho-alpha-D-ribose 1-diphosphate | I36E/M47D double mutant, kcat/KM: 0.0019 1/s*microM | Saccharolobus solfataricus | |
2.4.2.18 | additional information | - |
anthranilate | I36E/M47D double mutant, kcat/KM: 4.8 1/s*microM | Saccharolobus solfataricus | |
2.4.2.18 | additional information | - |
additional information | single mutants I36E and M47D show the same kcat as I36E/M47D double mutant within experimental error | Saccharolobus solfataricus | |
2.4.2.18 | additional information | - |
5-phospho-alpha-D-ribose 1-diphosphate | wild-type, kcat/KM: 0.0023 1/s*microM | Saccharolobus solfataricus | |
2.4.2.18 | additional information | - |
anthranilate | wild-type, kcat/KM: 4.8 1/s*microM | Saccharolobus solfataricus | |
2.4.2.18 | 0.28 | - |
anthranilate | +/-0.06, I36E/M47D double mutant, pH 7.2, 37°C, 0.01 microM subunit concentration | Saccharolobus solfataricus | |
2.4.2.18 | 0.41 | - |
anthranilate | +/-0.04, wild-type, pH 7.2, 37°C, 0.01 microM subunit concentration | Saccharolobus solfataricus |