Literature summary for 2.4.2.18 extracted from

Schwab, T.; Skegro, D.; Mayans, O.; Sterner, R.
A rationally designed monomeric variant of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus is as active as the dimeric wild-type enzyme but less thermostable (2008), J. Mol. Biol., 376, 506-516.

Cloned(Commentary)

Cloned (Comment)	Organism
wild-type and mutants in pQE40 for expression with N-terminal hexa-His tag in Escherichia coli W3110 trpEA2 (pDM)	Saccharolobus solfataricus

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants

Protein Variants	Comment	Organism
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
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
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

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.000058	-	anthranilate	I36E/M47D double mutant, pH 7.2, 37°C, 0.01 microM subunit concentration	Saccharolobus solfataricus
0.000085	-	anthranilate	wild-type, pH 7.2, 37°C, 0.01 microM subunit concentration	Saccharolobus solfataricus
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
0.18	-	5-phospho-alpha-D-ribose 1-diphosphate	wild-type, pH 7.2, 37°C, 0.01 microM subunit concentration	Saccharolobus solfataricus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	essential	Saccharolobus solfataricus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
32400	-	double mutant M47D/I36E, analytical-gel filtration chromatography	Saccharolobus solfataricus
34100	-	double mutant M47D/I36E, analytical ultracentrifugation (sedimentation equilibrium)	Saccharolobus solfataricus
38800	-	calculated for monomeric wild-type and mutants	Saccharolobus solfataricus
60100	-	mutant M47D, analytical ultracentrifugation (sedimentation equilibrium)	Saccharolobus solfataricus
61700	-	mutant M47D, analytical gel filtration chromatography	Saccharolobus solfataricus
63400	-	mutant I36E, analytical gel filtration chromatography	Saccharolobus solfataricus
64500	-	wild-type, analytical gel filtration chromatography	Saccharolobus solfataricus
66000	-	mutant I36E, analytical ultracentrifugation (sedimentation equilibrium)	Saccharolobus solfataricus
72200	-	wild-type, analytical ultracentrifugation (sedimentation equilibrium)	Saccharolobus solfataricus
77700	-	calculated for the dimeric wild-type and mutants	Saccharolobus solfataricus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
anthranilate + 5-phospho-alpha-D-ribose 1-diphosphate	Saccharolobus solfataricus	tryptophan biosynthesis, class III phosphoribosyltransfer	N-(5-phospho-D-ribosyl)-anthranilate + diphosphate	-	ir

Organism

Organism	UniProt	Comment	Textmining
Saccharolobus solfataricus	P50384	-	-

Purification (Commentary)

Purification (Comment)	Organism
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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
anthranilate + 5-phospho-alpha-D-ribose 1-diphosphate	tryptophan biosynthesis, class III phosphoribosyltransfer	Saccharolobus solfataricus	N-(5-phospho-D-ribosyl)-anthranilate + diphosphate	-	ir
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

Subunits

Subunits	Comment	Organism
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
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
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

Synonyms

Synonyms	Comment	Organism
AnPRT	-	Saccharolobus solfataricus
anthranilate phosphoribosyltransferase	-	Saccharolobus solfataricus
ssAnPRT	-	Saccharolobus solfataricus
ssTrpD	-	Saccharolobus solfataricus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	concentration-dependent kinetic stability of single mutants I36E and M47D during heat inactivation at 80°C	Saccharolobus solfataricus
additional information	-	dimerisation stabilizes against thermal denaturation	Saccharolobus solfataricus
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
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
83	-	mutant M47D, 12 microM, melting temperature at which half of the protein is unfolded deduced from DSC, pH 7.5	Saccharolobus solfataricus
90	-	mutant I36E, 12 microM, melting temperature at which half of the protein is unfolded deduced from DSC, pH 7.5	Saccharolobus solfataricus
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

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
additional information	-	5-phospho-alpha-D-ribose 1-diphosphate	I36E/M47D double mutant, kcat/KM: 0.0019 1/s*microM	Saccharolobus solfataricus
additional information	-	anthranilate	I36E/M47D double mutant, kcat/KM: 4.8 1/s*microM	Saccharolobus solfataricus
additional information	-	additional information	single mutants I36E and M47D show the same kcat as I36E/M47D double mutant within experimental error	Saccharolobus solfataricus
additional information	-	5-phospho-alpha-D-ribose 1-diphosphate	wild-type, kcat/KM: 0.0023 1/s*microM	Saccharolobus solfataricus
additional information	-	anthranilate	wild-type, kcat/KM: 4.8 1/s*microM	Saccharolobus solfataricus
0.28	-	anthranilate	+/-0.06, I36E/M47D double mutant, pH 7.2, 37°C, 0.01 microM subunit concentration	Saccharolobus solfataricus
0.41	-	anthranilate	+/-0.04, wild-type, pH 7.2, 37°C, 0.01 microM subunit concentration	Saccharolobus solfataricus