Literature summary for 2.4.2.19 extracted from

Liu, H.; Woznica, K.; Catton, G.; Crawford, A.; Botting, N.; Naismith, J.H.
Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from Homo sapiens (2007), J. Mol. Biol., 373, 755-763.

Cloned(Commentary)

Cloned (Comment)	Organism
in pEHISTEV (pBS-hQPRT as template) for expression in Escherichia coli BL21(DE3) or mutagenesis, expression with N-terminal hexa-His tag and TEV protease cleavage site, two extra N-terminal residues (Gly, Ala) remain after removal of hexa-His tag	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
PDB code: 2jbm (apo-hQPRTase), alpha/beta barrel fold (12 beta strands + 11 alpha helices) with N-terminal domain (residues 1-112, 279-291) and C-terminal domain (residues 113-278), similar to bacterial QPRTases (PDB: 1x1o), active site at alpha/beta open sandwich structure that faces an alpha/beta barrel of the adjacent subunit harbouring the quinolinic acid binding site (Arg102, Arg138, Arg161, Lys139, Lys171, pocket at the centre of the barrel), space group P2(1)2(1)2(1), unit-cell parameters: a = 111.5 A, b = 179.5 A, c = 194.7 A, 12 monomers in asymmetric unit arranged as 2 hexamers of D3 symmetry, sitting-drop vapour diffusion: 5 days, 20°C, 2 microlitre protein solution (10 mg/ml, pH7.5) + 2 microlitre precipitant (0.6 M potassium/sodium tartrate, 0.1 M sodium HEPES pH7.6), resolution of 2 A, phase determination using multiple wavelength anomalous diffraction on crystals of the Se-Met variant	Homo sapiens

Crystallization (Comment)

Organism

PDB code: 2jbm (apo-hQPRTase), alpha/beta barrel fold (12 beta strands + 11 alpha helices) with N-terminal domain (residues 1-112, 279-291) and C-terminal domain (residues 113-278), similar to bacterial QPRTases (PDB: 1x1o), active site at alpha/beta open sandwich structure that faces an alpha/beta barrel of the adjacent subunit harbouring the quinolinic acid binding site (Arg102, Arg138, Arg161, Lys139, Lys171, pocket at the centre of the barrel), space group P2(1)2(1)2(1), unit-cell parameters: a = 111.5 A, b = 179.5 A, c = 194.7 A, 12 monomers in asymmetric unit arranged as 2 hexamers of D3 symmetry, sitting-drop vapour diffusion: 5 days, 20°C, 2 microlitre protein solution (10 mg/ml, pH7.5) + 2 microlitre precipitant (0.6 M potassium/sodium tartrate, 0.1 M sodium HEPES pH7.6), resolution of 2 A, phase determination using multiple wavelength anomalous diffraction on crystals of the Se-Met variant

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
K139A	inactive, K139 is part of quinolinic acid binding site	Homo sapiens
K139S	inactive, K139 is part of quinolinic acid binding site	Homo sapiens
K171A	inactive, K171 is part of quinolinic acid binding site	Homo sapiens
K171S	inactive, K171 is part of quinolinic acid binding site	Homo sapiens
R102A	10% remaining activity, R102 is part of quinolinic acid binding site, denotes from the other subunit in the canonical dimer	Homo sapiens
R102Q	10% remaining activity, R102 is part of quinolinic acid binding site, denotes from the other subunit in the canonical dimer	Homo sapiens
R138Q	inactive, R138 is part of quinolinic acid binding site	Homo sapiens
R161A	20% remaining activity, R161 is part of quinolinic acid binding site and important for substrate binding	Homo sapiens
R161Q	inactive, R161 is part of quinolinic acid binding site	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism	Structure
5-phospho-alpha-D-ribose 1-diphosphate	substrate inhibition, mixed inhibition (competitive and non-competitive) above 0.3 mM 5-phospho-alpha-D-ribose 1-diphosphate	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0216	-	quinolinic acid	+/-0.003 mM, wild-type, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C, affinity to quinolinic acid is independent of 5-phospho-alpha-D-ribose 1-diphosphate concentration	Homo sapiens
0.0232	-	5-phospho-alpha-D-ribose 1-diphosphate	+/-0.0036 mM, wild-type, 0.3 mM quinolinic acid, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C, KM for 5-phospho-alpha-D-ribose 1-diphosphate increases with decreasing quinolinic acid concentration	Homo sapiens
0.319	-	quinolinic acid	+/-0.060 mM, mutant R161A, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate	Homo sapiens	type II phosphoribosyltransfer followed by irreversible decarboxylation, involved in catabolism of quinolinic acid and in NAD+ de novo synthesis	nicotinate D-ribonucleotide + diphosphate + CO2	-	ir

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q15274	-	-

Purification (Commentary)

Purification (Comment)	Organism
nickel affinity chromatography, TEV protease cleavage followed by nickel affinity chromatography, gel filtration, selenomethionine variant of hQPRTase purified in presence of 5 mM beta-mercaptoethanol	Homo sapiens

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	mutant R102A and R102Q, 10% remaining activity	Homo sapiens
additional information	-	mutant R161A, 20% remaining activity	Homo sapiens
0.09	-	wild-type, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, 30 min, 37°C, same order of magnitude as bacterial enzymes, 100% relative activity	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
additional information	no detectable activity for mutants R161Q, R138Q, K171A, K171S, K139A, K139S, 6 mM MgCl2, pH7.2, 37°C, 0.3 mM quinolinic acid, UV-based assay: 14 microgram purified enzyme, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 30 min	Homo sapiens	?	-	?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate	type II phosphoribosyltransfer followed by irreversible decarboxylation, involved in catabolism of quinolinic acid and in NAD+ de novo synthesis	Homo sapiens	nicotinate D-ribonucleotide + diphosphate + CO2	-	ir
quinolinic acid + 5-phospho-alpha-D-ribose 1-diphosphate	6 mM MgCl2, pH7.2, 37°C, 0.3 mM quinolinic acid, HPLC-based assay: 7 microgram purified enzyme, 1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 20 min, UV-based assay: 14 microgram purified enzyme, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 30 min	Homo sapiens	nicotinic acid mononucleotide + diphosphate + CO2	HPLC-based assay: 254 nm (quantification of nicotinic acid mononucleotide and quinolinic acid), UV-based assay: 266 nm (quantification of nicotinic acid mononucleotide)	ir

Subunits

Subunits	Comment	Organism
hexamer	according to gel filtration, 3 canonical (AB) dimers (formed by two-fold rotation placing N-terminus of monomer A next to C-terminus of monomer B) related to each other by 3-fold rotation axis, hexameric D3 symmetry, active sites in close proximity at interface between monomers A and monomers B	Homo sapiens

Synonyms

Synonyms	Comment	Organism
hQPRTase	human QPRTase	Homo sapiens
QPRTase	-	Homo sapiens
quinolinate phosphoribosyltransferase	-	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
additional information	-	quinolinic acid	Vmax = 0.71(+/-0.0) microM/min, mutant R161A, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm	Homo sapiens
additional information	-	5-phospho-alpha-D-ribose 1-diphosphate	Vmax = 0.93(+/-0.03) microM/min, wild-type, 0.3 mM quinolinic acid, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm, Vmax increases with increasing 5-phospho-alpha-D-ribose 1-diphosphate concentrations up to 5 mM	Homo sapiens
additional information	-	quinolinic acid	Vmax = 1.19(+/-0.05) microM/min, wild-type, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, UV-based assay measuring nicotinic acid mononucleotide formation at 266 nm	Homo sapiens