Literature summary for 2.4.2.18 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.

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
D83G/F149S	activity increases up to about 2-5 mM MgCl2 and decreases only moderately at higher Mg2+ concentrations	Saccharolobus solfataricus
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

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

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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
0.00002	-	anthranilate	wild-type, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C	Saccharolobus solfataricus
0.00012	-	anthranilate	mutant D83G, in the presence of 2 mM MgCl2, 50 mM Tris-HCl, pH 7.2, at 37°C	Saccharolobus solfataricus
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
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
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
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
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
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
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
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
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

Organism	UniProt	Comment	Textmining
Saccharolobus solfataricus	P50384	-	-

Purification (Commentary)

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

Substrates and Products (Substrate)

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

Synonyms

Synonyms	Comment	Organism
AnPRT	-	Saccharolobus solfataricus
TrpD	-	Saccharolobus solfataricus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
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]

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