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Literature summary extracted from

  • Schlee, S.; Deuss, M.; Bruning, M.; Ivens, A.; Schwab, T.; Hellmann, N.; Mayans, O.; Sterner, R.
    Activation of anthranilate phosphoribosyltransferase from Sulfolobus solfataricus by removal of magnesium inhibition and acceleration of product release (2009), Biochemistry, 48, 5199-5209.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.2.18 amplified fragments containing the full-length trpD gene ligated into the pQE40 vector, transformed into Escherichia coli competent DH5alpha cells. Wild-type and its mutants expressed heterologously in Escherichia coli strain W3110 trpEA2trpEA2, containing the helper plasmid pDM,1 Saccharolobus solfataricus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.2.18 mutant D83G/F149S in complex with 5-phospho-alpha-D-ribose 1-diphosphate and Mn2+, to 2.25 A resolution. Protein backbone of mutant D83G/F149S shows no detectable differences to the wild-type enzyme, whereas 5-phospho-alpha-D-ribose 1-diphosphate bound to mutant D83G/F149S adopts an extended conformation that contrasts markedly with the S compact shape observed in complexes of the wild-type enzyme Saccharolobus solfataricus

Protein Variants

EC Number Protein Variants Comment Organism
2.4.2.18 D83G inhibition by Mg2+ only at very high concentrations, alters the binding mode of the substrate Mg2+-5-phospho-alpha-D-ribose 1-diphosphate Saccharolobus solfataricus
2.4.2.18 D83G/F149S activity increases up to about 2-5 mM MgCl2 and decreases only moderately at higher Mg2+ concentrations Saccharolobus solfataricus
2.4.2.18 F149S is inhibited by MgCl2 to a similar extent as wild-type, facilitates product release by increasing the conformational flexibility of the enzyme Saccharolobus solfataricus

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.4.2.18 Mg2+ marked inhibition at MgCl2 concentrations higher than 0.1 mM Saccharolobus solfataricus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.2.18 0.000018
-
 anthranilate wild-type, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.00002
-
 anthranilate wild-type, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.00012
-
 anthranilate mutant D83G, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.0012
-
 anthranilate mutant F149S, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.0031
-
 anthranilate mutant D83G/F149S, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.0036
-
 anthranilate mutant D83G/F149S, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.013
-
 5-phospho-alpha-D-ribose 1-diphosphate mutant D83G/F149S, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.023
-
 5-phospho-alpha-D-ribose 1-diphosphate mutant D83G/F149S, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.038
-
 5-phospho-alpha-D-ribose 1-diphosphate mutant D83G, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.047
-
 5-phospho-alpha-D-ribose 1-diphosphate wild-type, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.151
-
 5-phospho-alpha-D-ribose 1-diphosphate mutant F149S, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 1.54
-
 5-phospho-alpha-D-ribose 1-diphosphate wild-type, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus

Organism

EC Number Organism UniProt Comment Textmining
2.4.2.18 Saccharolobus solfataricus P50384
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.2.18 wild-type and mutants purified by heat precipitation and metal chelate affinity chromatography Saccharolobus solfataricus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.2.18 anthranilate + 5-phospho-alpha-D-ribose 1-diphosphate
-
 Saccharolobus solfataricus N-(5-phospho-D-ribosyl)-anthranilate + diphosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.4.2.18 AnPRT
-
 Saccharolobus solfataricus
2.4.2.18 TrpD
-
 Saccharolobus solfataricus

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.4.2.18 90
-
 wild-type protein and mutant D83G show melting temperatures of 90 and 91°C, respectively, mutant F149S protein and the double mutant D83G/F149S exhibit melting temperature values of 82°C Saccharolobus solfataricus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.2.18 0.24
-
 anthranilate wild-type, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 0.33
-
 anthranilate wild-type, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 1.2
-
 anthranilate mutant F149S, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 2
-
 anthranilate mutant D83G, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 4.4
-
 anthranilate mutant D83G/F149S, in the presence of 0.05 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus
2.4.2.18 13.3
-
 anthranilate mutant D83G/F149S, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C Saccharolobus solfataricus