Information on EC 2.4.2.19 - nicotinate-nucleotide diphosphorylase (carboxylating)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY
2.4.2.19
-
RECOMMENDED NAME
GeneOntology No.
nicotinate-nucleotide diphosphorylase (carboxylating)
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
beta-nicotinate D-ribonucleotide + diphosphate + CO2 = pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
show the reaction diagram
-
-
-
-
beta-nicotinate D-ribonucleotide + diphosphate + CO2 = pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
show the reaction diagram
formation of a ternary complex consisting the enzyme, pyridine-2,3-dicarboxylate and 5-phospho-alpha-D-ribose 1-diphosphate
-
beta-nicotinate D-ribonucleotide + diphosphate + CO2 = pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
show the reaction diagram
ordered binding mechanism
P30011
beta-nicotinate D-ribonucleotide + diphosphate + CO2 = pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
show the reaction diagram
formation of a ternary complex comprising the enzyme, quinolinate and 5-phosphoribosyl-1-diphosphate
-
beta-nicotinate D-ribonucleotide + diphosphate + CO2 = pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
show the reaction diagram
predominantly ordered kinetic mechanism in which productive binding of quinolinate precedes that of 5-phospho-alpha-D-ribose 1-diphosphate
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pentosyl group transfer
-
-
-
-
pentosyl group transfer
-
-
PATHWAY
KEGG Link
MetaCyc Link
Metabolic pathways
-
NAD biosynthesis from 2-amino-3-carboxymuconate semialdehyde
-
NAD biosynthesis I (from aspartate)
-
Nicotinate and nicotinamide metabolism
-
nicotine biosynthesis
-
superpathway of nicotine biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
nicotinate-D-ribonucleotide:diphosphate phospho-alpha-D-ribosyltransferase (carboxylating)
The reaction is catalysed in the opposite direction. Since quinolinate is synthesized from L-tryptophan in eukaryotes, but from L-aspartate in some prokaryotes, this is the first NAD+ biosynthesis enzyme shared by both eukaryotes and prokaryotes [3].
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
general stress protein 70
-
-
-
-
GSP70
-
-
-
-
Hp-QAPRTase
-
-
hQPRTase
Q15274
human QPRTase
NAD pyrophosphorylase
-
-
-
-
NadC
-
-
-
-
NadC
Q9X1X8
-
nicotinate mononucleotide pyrophosphorylase (carboxylating) (EC 2.4.2.19)
-
-
-
-
nicotinate-nucleotide pyrophosphorylase (carboxylating)
-
-
-
-
nicotinate-nucleotide:pyrophosphate phospho-alpha-D-ribosyltransferase (decarboxylating)
-
-
-
-
pyrophosphorylase, nicotinate mononucleotide (carboxylating)
-
-
-
-
QAPRTase
-
-
-
-
QAPRTase
-
-
QAPRTase
Burkholderia sp. PsJN
-
-
-
QAPRTase
P43619
-
QAPRTase
-
-
QAPRTase
Q9X1X8
-
QPRTase
-
-
QPRTase
Q15274
-
QPRTase
-
-
-
-
QPT1
Q0WX55
-
QPT2
Q0WX54
-
quinolate phosphoribosyltransferase
-
-
quinolinate phosphoribosyl transferase
P43619
-
quinolinate phosphoribosyltransferase
-
-
-
-
quinolinate phosphoribosyltransferase
-
-
quinolinate phosphoribosyltransferase
Q15274
-
quinolinate phosphoribosyltransferase
-
-
quinolinate phosphoribosyltransferase
-
-
quinolinate phosphoribosyltransferase (decarboxylating)
-
-
-
-
quinolinate phosphoribosyltransferase 1
Q0WX55
-
quinolinate phosphoribosyltransferase 2
Q0WX54
-
quinolinate phosphoribosyltransferase [decarboxylating]
-
-
-
-
quinolinic acid phosphoribosyltransferase
-
-
-
-
quinolinic phosphoribosyltransferase
-
-
-
-
TM1645
Q9X1X8
-
type II quinolic acid phosphoribosyltransferase
Q9X1X8
-
type II quinolinic acid phosphoribosyltransferase
-
-
CAS REGISTRY NUMBER
COMMENTARY
37277-74-0
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
DOB1 and other phthalate-degrading strains have two dissimilar genes for this enzyme, while non-phthalate-degrading strains have only a single gene
-
-
Manually annotated by BRENDA team
strain PsJN
-
-
Manually annotated by BRENDA team
Burkholderia sp. PsJN
strain PsJN
-
-
Manually annotated by BRENDA team
nov. subsp. quinolinicus
-
-
Manually annotated by BRENDA team
subsp. quinolinicus
-
-
Manually annotated by BRENDA team
strain MSB8, gene TM1645
SwissProt
Manually annotated by BRENDA team
i.e. Vigna radiata
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
overexpression and purification of the mutant enzymes produce 200-250 mg of protein/6 liter of culture similar to that of the wild-type enzyme
physiological function
-
interacts with active-caspase-3, depletion of quinolinate phosphoribosyltransferase protein increases intracellular caspase-3 activity, quinolinate phosphoribosyltransferase-depleted cells are highly sensitive to spontaneous cell death, quinolinate phosphoribosyltransferase protein prevents spontaneous cell death through inhibition of overproduction of active-caspase-3, identified as a caspase-3 binding protein, not a substrate for caspase-3
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O
beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate
show the reaction diagram
-
-
-
-
?
nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O
beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate
show the reaction diagram
-
-
-
-
-
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
P30011
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
-
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
ir
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Q9X1X8, -
-
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
-
i.e. beta-niacin mononucleotide
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Q9X1X8, -
enzyme provides the de novo source of nicotinate mononucleotide
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
synthesis of precursors for trigonelline biosynthesis, feeding experiments, overview, direct conversion of quinolinic acid to nicotinic acid mononucleotide
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
O25909
essential enzyme in the NAD+ biosynthetic pathway
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Q15274
type II phosphoribosyltransfer followed by irreversible decarboxylation, involved in catabolism of quinolinic acid and in NAD+ de novo synthesis
-
-
ir
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate + H+
beta-nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Burkholderia sp., Burkholderia sp. PsJN
-
Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN
-
-
?
quinolinic acid + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinic acid mononucleotide + diphosphate + CO2
show the reaction diagram
P43619, -
-
-
-
?
quinolinic acid + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinic acid mononucleotide + diphosphate + CO2
show the reaction diagram
Q15274
6 mM MgCl2, pH7.2, 37C, 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
HPLC-based assay: 254 nm (quantification of nicotinic acid mononucleotide and quinolinic acid), UV-based assay: 266 nm (quantification of nicotinic acid mononucleotide)
-
ir
quinolinic acid + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinic acid mononucleotide + diphosphate + CO2
show the reaction diagram
-
Arg175 is essential for catalysis, critical role for Lys185 in the chemistry of the QAPRTase intermediate, interfacial role for Arg118 during catalysis
-
-
?
quinolinic acid + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinic acid mononucleotide + diphosphate + CO2
show the reaction diagram
-
Lys153 is necessary for catalysis and important for 5-phospho-alpha-D-ribose 1-diphosphate binding, Glu214 provides a hydrogen bond necessary for catalysis but does not act as a base or electrostatically to stabilize the transition state, Lys284 is involved in 5-phospho-alpha-D-ribose 1-diphosphate binding, and Asp235 is not essential
-
-
?
additional information
?
-
-
key enzyme of NAD+ biosynthesis
-
-
-
additional information
?
-
-
enzyme of the kynurenine pathway
-
-
-
additional information
?
-
-
key enzyme in NAD+ biosynthesis, also plays an important role in ensuring nicotinic acid available for the synthesis of defensive pyridine alkaloids
-
-
-
additional information
?
-
-
intermediary enzyme in the de novo NAD biosynthetic pathway
-
-
-
additional information
?
-
-
the recruitment of this gene for growth on phthalate gives Burkholderia cepacia an advantage over other phthalate-degrading bacteria in the environment
-
-
-
additional information
?
-
-
key enzyme in catabolism of quinolinate. Quinolinate acts as a most potent endogenous exitotoxin to neurons. Elevation of quinolinate levels in the brain has been linked to the pathogenesis of neurodegenerative disorders
-
-
-
additional information
?
-
-
essential enzyme for the de novo biosynthesis of NAD+
-
-
-
additional information
?
-
-
enzyme is probably involved in the regulation of nicotine biosynthesis
-
-
-
additional information
?
-
Q15274
no detectable activity for mutants R161Q, R138Q, K171A, K171S, K139A, K139S, 6 mM MgCl2, pH7.2, 37C, 0.3 mM quinolinic acid, UV-based assay: 14 microgram purified enzyme, 0.1 mM 5-phospho-alpha-D-ribose 1-diphosphate, 30 min
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Q9X1X8, -
enzyme provides the de novo source of nicotinate mononucleotide
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
-
synthesis of precursors for trigonelline biosynthesis, feeding experiments, overview
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
O25909
essential enzyme in the NAD+ biosynthetic pathway
-
-
?
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate
nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Q15274
type II phosphoribosyltransfer followed by irreversible decarboxylation, involved in catabolism of quinolinic acid and in NAD+ de novo synthesis
-
-
ir
pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate + H+
beta-nicotinate D-ribonucleotide + diphosphate + CO2
show the reaction diagram
Burkholderia sp., Burkholderia sp. PsJN
-
Burkholderia sp. strain PsJN stimulates root growth of potato explants compared to uninoculated controls under gnotobiotic conditions. QAPRTase may play a role in the signal pathway for promotion of plant growth by PsJN
-
-
?
additional information
?
-
-
key enzyme of NAD+ biosynthesis
-
-
-
additional information
?
-
-
enzyme of the kynurenine pathway
-
-
-
additional information
?
-
-
key enzyme in NAD+ biosynthesis, also plays an important role in ensuring nicotinic acid available for the synthesis of defensive pyridine alkaloids
-
-
-
additional information
?
-
-
intermediary enzyme in the de novo NAD biosynthetic pathway
-
-
-
additional information
?
-
-
the recruitment of this gene for growth on phthalate gives Burkholderia cepacia an advantage over other phthalate-degrading bacteria in the environment
-
-
-
additional information
?
-
-
key enzyme in catabolism of quinolinate. Quinolinate acts as a most potent endogenous exitotoxin to neurons. Elevation of quinolinate levels in the brain has been linked to the pathogenesis of neurodegenerative disorders
-
-
-
additional information
?
-
-
essential enzyme for the de novo biosynthesis of NAD+
-
-
-
additional information
?
-
-
enzyme is probably involved in the regulation of nicotine biosynthesis
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
NAD+
-
0.1 mM, 75% inhibition
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Cd2+
-
can partially replace Mg2+
Co2+
-
can partially replace Mg2+
K+
-
monovalent cation required, K+, Li+ or NH4+ markedly stimulate
Li+
-
monovalent cation required, K+, Li+ or NH4+ markedly stimulate
Mg2+
-
maximal activity at 0.1-0.2 mM
Mg2+
-
divalent cation required, Km: 0.2 mM
Mg2+
-
optimal Mg2+ concentration: 1 mM
Mg2+
-
absolute requirement. Optimal concentration is 1 mM in absence of ATP and 7 mM in presence of ATP at 5 mM
Mg2+
-
absolute requirement
Mg2+
P30011
required
Mg2+
-
divalent cation required, Mg2+ is most effective at 1 mM
Mg2+
-
absolute requirement for divalent cations. Mg2+ is most effective. Optimal concentrations are 4, 6 and 10 mM for 20, 40 and 100 mM sodium acetate/acetic acid buffer, pH 5.5
Mg2+
-
absolute requirement for a divalent metal ion, Mg2+ is most effective, optimal activity at 1.5 mM
Mg2+
-
-
Mn2+
-
half as effective as Mg2+
Mn2+
-
0.3 mM, can fully replace Mg2+
Mn2+
-
at 1 mM, 80% of the activation with Mg2+
NH4+
-
monovalent cation required, K+, Li+ or NH4+ markedly stimulate
Zn2+
-
can partially replace Mg2+
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2-Hydroxynicotinate
-
50% at 0.01 M
2-oxoglutarate
-
1 mM, 60% inhibition
2-oxoglutarate
-
1 mM, 55% inhibition
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
5-phosphoribosyl-1-diphosphate
-
inhibition at alkaline pH and at physiological pH, pH 7.4, but not at an acidic pH, competitive for quinolinate. In presence of 30% glycerol, both the kidney and liver enzyme are inhibited, even at acidic pH
5-phosphoribosyl-1-diphosphate
-
-
acetic acid
-
26% inhibition
ADP
-
2 mM, 25% inhibition
ADP
-
1 mM, 12% inhibition
Ag+
-
1 mM, complete inhibition
Al3+
-
1 mM, 82.8% inhibition
AMP
-
2 mM, 7% inhibition
aspartic acid
-
1 mM, 23% inhibition
aspartic acid
-
1 mM, 37% inhibition
ATP
-
2 mM, 82% inhibition
ATP
-
2 mM, 82% inhibition; inhibition is removed by raising Mg2+ concentrations
ATP
-
1 mM, 22% inhibition; inhibition is completely recovered by raising Mg2+ concentration
Ba2+
-
1 mM, 38.1% inhibition
Ca2+
-
1 mM, 34.9% inhibition
Cd2+
-
1 mM, 75.5% inhibition
CDP
-
2 mM, 20% inhibition
CDP
-
1 mM, 13% inhibition
Citric acid
-
1 mM, 95% inhibition
Citric acid
-
1 mM, 89% inhibition
Co2+
-
1 mM, 65.2% inhibition
Cr3+
-
1 mM, 46.4% inhibition
CTP
-
2 mM, 77% inhibition
CTP
-
1 mM, 30% inhibition
Cu2+
-
1 mM, 87% inhibition
Cu2+
-
1 mM, 86.9% inhibition
D-fructose-1,6-diphosphate
P30011
competitive with respect to 5-phosphoribosyl-1-diphosphate and noncompetitive with respect to quinolinate
diphosphate
P30011
noncompetitive with respect to both 5-phosphoribosyl-1-diphosphate and quinolinate
dipicolinic acid
-
1 mM, 24% inhibition
dithiobis(2-nitrobenzoic acid)
-
10 mM, 88.7% inhibition
dTDP
-
1 mM, 14% inhibition
dTTP
-
1 mM, 29% inhibition
Fe2+
-
1 mM, 93% inhibition
-
Fe2+
-
1 mM, 61.5% inhibition
-
Fe3+
-
1 mM, 92.9% inhibition
-
formic acid
-
1 mM, 54% inhibition
Fumaric acid
-
1 mM, 72% inhibition
Fumaric acid
-
1 mM, 67% inhibition
GDP
-
2 mM, 29% inhibition
glycerol
-
inhibition increases as the pH raises
glycerol
-
glycerol markedly activates enzyme activity at pH 6.1 and 6.5, inhibition above pH 7.0, inhibition is strongest at pH 9.0
GTP
-
2 mM, 77% inhibition
GTP
-
1 mM, 17% inhibition
Hg2+
-
1 mM, 99.9% inhibition
IDP
-
2 mM, 29% inhibition
IMP
-
2 mM, 6% inhibition
isocinchomeric acid
-
1 mM, 15% inhibition
ITP
-
2 mM, 81% inhibition
ITP
-
1 mM, 24% inhibition
L-Glutamic acid
-
1 mM, 31% inhibition
L-Glutamic acid
-
1 mM, 41% inhibition
L-Malic acid
-
1 mM, 75% inhibition
L-Malic acid
-
1 mM, 67% inhibition
Lactic acid
-
1 mM, 29% inhibition
lutidinic acid
-
1 mM, 17% inhibition
Maleic acid
-
1 mM, 34% inhibition
Maleic acid
-
1 mM, 42% inhibition
methyl-3-amidopyridine-2-carboxylate
-
-
methyl-3-cyanopyridine 2-carboxylate
-
-
Mg2+
-
1 mM, 7.7% inhibition
Mn2+
-
1 mM, 80.3% inhibition
Monoiodoacetic acid
-
5 mM, 50% inhibition
Na4P2O7
-
3 mM, complete inhibition
NAD+
-
19% inhibition at 1 mM, 15% inhibition at 10 mM
NEM
-
5 mM, 86% inhibition
Ni2+
-
1 mM, 76.5% inhibition
nicotinate mononucleotide
P30011
competitive with respect to 5-phosphoribosyl-1-diphosphate
nicotinate mononucleotide
-
1 mM, 46% inhibition
oxaloacetic acid
-
1 mM, 56% inhibition
oxaloacetic acid
-
1 mM, 59% inhibition
PCMB
-
0.05 mM, complete inhibition
Phthalic acid
-
competitive to quinolinate
Phthalic acid
P30011
dead-end inhibitor, competitive with respect to quinolinate, uncompetitive with respect to 5-phosphoribosyl-1-diphosphate
Phthalic acid
-
-
Picolinic acid
-
-
Sr2+
-
47.6% inhibition
succinic acid
-
1 mM, 51% inhibition
succinic acid
-
1 mM, 46% inhibition
UDP
-
2 mM, 32% inhibition
UTP
-
2 mM, 74% inhibition
UTP
-
1 mM, 19% inhibition
Zn2+
-
1 mM, 82.1% inhibition
Monoiodoacetic acid
-
50 mM, 99.8% inhibition
additional information
-
nicotinic acid and nicotinamide inhibit growth of roots
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
glycerol
-
glycerol markedly activates enzyme activity at pH 6.1 and pH 6.5, inhibition above pH 7.0, inhibition is strongest at pH 9.0
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0156
-
5-phospho-alpha-D-ribose 1-diphosphate
P30011
-
0.021
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.022
-
5-phospho-alpha-D-ribose 1-diphosphate
-
enzyme from liver
0.023
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.023
-
5-phospho-alpha-D-ribose 1-diphosphate
-
enzyme from brain
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, 37C, KM for 5-phospho-alpha-D-ribose 1-diphosphate increases with decreasing quinolinic acid concentration
0.03
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.032
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.0322
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.045
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.05
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.057
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.057
-
5-phospho-alpha-D-ribose 1-diphosphate
-
wild-type
0.057
-
5-phospho-alpha-D-ribose 1-diphosphate
-
wild-type, at 25C
0.111
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.128
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.128
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant E214Q
0.14
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.18
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.547
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant H188A, at 25C
0.55
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant E214D
0.604
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant E214A
0.832
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant R152A, at 25C
1.138
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant D235A
2.181
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant K185A, at 25C
3.138
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant R118A, at 25C
4.12
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant K284A
0.01
-
nicotinic acid
-
-
0.151
-
nicotinic acid
-
mutant K185A, at 25C
0.0051
-
Quinolinate
-
-
0.0056
-
Quinolinate
-
-
0.0064
-
Quinolinate
P30011
-
0.011
-
Quinolinate
-
-
0.012
-
Quinolinate
-
-
0.012
-
Quinolinate
-
enzyme from liver and brain
0.04
-
Quinolinate
-
-
0.06
-
Quinolinate
-
-
0.091
-
Quinolinate
-
-
0.12
-
Quinolinate
-
-
0.133
-
Quinolinate
-
-
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, 37C, affinity to quinolinic acid is independent of 5-phospho-alpha-D-ribose 1-diphosphate concentration
0.027
-
Quinolinic acid
-
wild-type, at 25C
0.051
-
Quinolinic acid
-
in presence of 30% glycerol
0.067
-
Quinolinic acid
-
mutant H188A, at 25C
0.136
-
Quinolinic acid
-
mutant R118A, at 25C
0.278
-
Quinolinic acid
-
mutant R152A, at 25C
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, 37C
1.478
-
Quinolinic acid
-
mutant K185A, at 25C
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00024
-
Quinolinic acid
-
mutant R118A, at 25C
0.0016
-
Quinolinic acid
-
mutant K185A, at 25C
0.03
-
Quinolinic acid
-
mutant R152A, at 25C
1
-
Quinolinic acid
-
mutant H188A, at 25C; wild-type, at 25C
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
additional information
-
additional information
-
turnover rate is decreased by adding glycerol
-
additional information
-
additional information
-
-
-
0.048
-
nicotinic acid
-
mutant K185A, at 25C
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; 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
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00004
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant E214A
5436
0.000049
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant E214D
5436
0.000064
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant R118A, at 25C
5436
0.0007
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant K284A
5436
0.00073
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant K185A, at 25C
5436
0.035
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant R152A, at 25C
5436
1.8
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant H188A, at 25C
5436
3.9
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant E214Q
5436
12
-
5-phospho-alpha-D-ribose 1-diphosphate
-
mutant D235A
5436
18
-
5-phospho-alpha-D-ribose 1-diphosphate
-
wild-type
5436
18
-
5-phospho-alpha-D-ribose 1-diphosphate
-
wild-type, at 25C
5436
0.32
-
nicotinic acid
-
mutant K185A, at 25C
14511
0.0011
-
Quinolinic acid
-
mutant K185A, at 25C
16108
0.0015
-
Quinolinic acid
-
mutant R118A, at 25C
16108
0.0019
-
Quinolinic acid
-
mutant E214A; mutant E214D
16108
0.023
-
Quinolinic acid
-
mutant K284A
16108
0.1
-
Quinolinic acid
-
mutant R152A, at 25C
16108
12
-
Quinolinic acid
-
mutant E214Q
16108
16
-
Quinolinic acid
-
mutant H188A, at 25C
16108
37
-
Quinolinic acid
-
wild-type
16108
37
-
Quinolinic acid
-
wild-type, at 25C
16108
52
-
Quinolinic acid
-
mutant D235A
16108
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.05
-
5-phosphoribosyl-1-diphosphate
-
enzyme from liver
0.265
-
5-phosphoribosyl-1-diphosphate
-
-
0.8
-
5-phosphoribosyl-1-diphosphate
-
enzyme from kidney
0.0838
-
nicotinate mononucleotide
-
-
0.0014
-
Phthalic acid
-
-
0.17
-
Phthalic acid
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.0006
-
-
wild-type with nicotinic acid at 1 mM and 2 mM 5-phospho-alpha-D-ribose 1-diphosphate
0.01235
-
-
-
0.0133
-
-
enzyme from liver
0.0187
-
-
enzyme from brain
0.0227
0.0919
-
enzyme from kidney
0.05169
-
-
-
0.0517
-
-
-
0.07
-
-
mutant R152A with nicotinic acid at 1 mM and 2 mM 5-phospho-alpha-D-ribose 1-diphosphate
0.073
-
-
-
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, 37C, same order of magnitude as bacterial enzymes, 100% relative activity
0.091
-
-
-
0.1
-
-
mutant R152A with quinolinic acid at 1 mM and 2 mM 5-phospho-alpha-D-ribose 1-diphosphate
0.88
-
-
-
1.2
-
-
wild-type with quinolinic acid at 1 mM and 2 mM 5-phospho-alpha-D-ribose 1-diphosphate
additional information
-
P30011
-
additional information
-
-
-
additional information
-
-
mutant R102A and R102Q, 10% remaining activity; mutant R161A, 20% remaining activity
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
-
-
in presence of 1 mM 5-phospho-alpha-D-ribose 1-diphosphate, enzyme from kidney
5.5
-
-
-
6
-
-
in presence of 1 mM 5-phospho-alpha-D-ribose 1-diphosphate, enzyme from liver
6.2
-
-
-
6.5
7.7
-
-
6.5
-
-
-
9
-
-
in presence of 0.4 mM 5-phospho-alpha-D-ribose 1-diphosphate, enzyme from kidney and liver
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.2
7.8
-
half maximal activity at pH 5.2 and pH 7.8
6.4
7.3
-
pH 6.4: about 50% of maximal activity, pH 7.3: about 55% of maximal activity
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.1
-
Q9X1X8, -
sequence calculation, residues 1-273
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
embryonic axes, high enzyme activity
Manually annotated by BRENDA team
-
endosperm of etiolated seedlings
Manually annotated by BRENDA team
-
transcript level increases markedly 12-24 h after damage to aerial tissue
Manually annotated by BRENDA team
-
transcript level increases markedly 12-24 h after damage to aerial tissue
Manually annotated by BRENDA team
-
endosperm of etiolated seedlings
Manually annotated by BRENDA team
additional information
-
nicotinic acid metabolism and content of trigonelline in different tissues of the seeds and seedlings and during differentiation
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Burkholderia pseudomallei (strain 1710b)
Ehrlichia chaffeensis (strain ATCC CRL-10679 / Arkansas)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
68000
-
-
gel filtration
70000
-
P30011
gel filtration
72000
-
-
sucrose density gradient centrifugation
160000
-
-
gel filtration
160000
-
-
sucrose density gradient centrifugation, gel filtration
167000
-
-
sucrose density gradient centrifugation
170000
-
-
gel filtration
172000
-
-
sedimetation velocity method
173000
-
-
gel filtration
178000
-
-
calculation from sedimentation and ultracentrifugation data
202000
-
-
equilibrium sedimentation
210000
-
-
gel filtration
210000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 33500, SDS-PAGE; x * 34200, equilibrium sedimentation in guanidine HCl
?
-
x * 34000, SDS-PAGE
?
-
x * 33500, SDS-PAGE
?
Q9X1X8, -
x * 30048, crystal structure, residues 1-273
dimer
-
2 * 35000, SDS-PAGE
dimer
P30011
2 * 36000, SDS-PAGE
dimer
-
-
dimer
-
wild-type and mutants, gel filtration
hexamer
O25909
Hp-QAPRTase forms a hexamer with a - trimer of dimers - configuration
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
hexamer
P43619, -
6 * 191000, dynamic light scattering analysis
homohexamer
-
gel filtration
homohexamer
-
gel filtration
octamer
-
8 * 27500, SDS-PAGE
pentamer
-
5 * 32000, SDS-PAGE
pentamer
-
5 * 34000, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
glycoprotein
-
enzyme contains 1% mannose
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
crystal structure of Hp-QAPRTase with bound quinolinic acid, nicotinic acid mononucleotide, and phthalic acid. Hp-QAPRTase crystals are grown at 20C using the hanging drop vapor diffusion method
O25909
hanging-drop vapor-diffusion method
-
hanging-drop vapour-diffusion method, PEG MME 2K
-
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, 20C, 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
-
hangig-drop vapor diffusion method, X-ray crystal structure of the apoenzyme is determined by multiple isomorphous replacement at 2.4 A resolution, complex with quinolinate, phthalate, nicotinate mononucleotideand ternary complex with phthalate and a substrate analog 5-phosphoribosyl-1-(beta-methylene)diphosphate
-
apo, quinolinate-bound, 5-phospho-alpha-D-ribose 1-diphosphate-bound, and phthalate-bound forms. Crystallized at room temperature by the hanging drop vapor diffusion method. Both apo and holo crystals belong to space group R32 (alpha = 90, beta = 90, and gamma = 120). One molecule per asymmetric unit except in QAPRTase holophthalate crystals where two molecules are found. The unit cell dimensions are: a = b = 154.9 A and c = 68.9 A (apo), a = b = 154.8 A and c = 68.6 A (holoquinolinate), a = b = 154.9 A and c = 70.6 A (holo-5-phospho-alpha-D-ribose 1-diphosphate), a = b = 155.5 A and c = 121.1 A (holophthalate), a = b = 154.7 A and c = 69.3 A (holophthalate-5-phospho-alpha-D-ribose 1-diphosphate)
P43619, -
hanging-drop vapor-diffusion method, determination of crystal structure of the enzyme with bound quinolinate to 2.8 A resolution and with bound nicotinic acid mononucleotide
-
hanging-drop vapour-diffusion method, PEG 8000
-
purified recombinant enzyme, hanging drop vapour diffusion method, 10 mg/ml protein, crystallization solution contains 10% PEG 6000, and 0.1 M MES, pH 6.0, X-ray diffraction structure determination and analysis at 2.5 A resolution, molecular replacement, model construction
Q9X1X8, -
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
3
3.5
-
37C, denatured abruptly
4.5
9.5
-
37C, 30 min, completely stable
5.5
10
-
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
55
-
-
10 min, 48% loss of activity
80
-
-
3 min, 50% loss of activity
additional information
-
-
quinolinate at 0.8 mM gives 50% protection against heat inactivation. 22% inhibition in presence of quinolinate
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4C, stable for 1 month
P30011
-20C, enzyme in treated extract, stable
-
-90C, stable for several months in 0.05 M potassium phosphate buffer, pH 7.0, 50% sucrose w/v and 0.01 M dithiothreitol
-
0-4C, crystalline enzyme is stable for at least 2 years
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant enzyme
-
ammonium sulfate precipitation, dye ligand chromatography (Blue-Sepharose), ion exchange chromatography
-
immobilized metal ion affinity chromatography (Ni2+), gel filtration
-
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
-
by anion exchange chromatography
P43619, -
mutants purified by gel filtration, to homogeneity
-
mutants purified to homogeneity in 250 mg yield from a 6 liter culture, by gel filtration
-
from porcine kidney, ammonium sulfate fractionation, ion exchange chromatography (DEAE), gel filtration
-
recombinant N-terminally His-tagged enzyme from Escherichia coli b nickel affinity chromatography
Q9X1X8, -
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Escherichia coli as glutathione S-transferase fusion protein
-
expressed in Escherichia coli BL21(DE3)-RIPL Codon Plus
-
expression in Escherichia coli
-
His-tagged protein expressed in Escherichia coli C41 (DE3)
-
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
-
overexpression in Escherichia coli
-
expression in Escherichia coli
-
expressed in Escherichia coli TH265 (nadC mutant)(complementation approach); expressed in Escherichia coli TH265 (nadC mutant)(complementation approach)
Q0WX54, Q0WX55
expression in Escherichia coli
-
into plasmid pTYB12 and expressed in Escherichia coli BL21
P43619, -
nadC coding sequence cloned into the pRSETC expression vector, overexpressed in Escherichia coli nadC-deleted host strain ZB100
-
subcloned into a T7-based expression system
-
gene TM1645, expression of N-terminally His-tagged enzyme in Escherichia coli
Q9X1X8, -
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
K139A
-
inactive, K139 is part of quinolinic acid binding site
K139S
-
inactive, K139 is part of quinolinic acid binding site
K171A
-
inactive, K171 is part of quinolinic acid binding site
K171S
-
inactive, K171 is part of quinolinic acid binding site
R102A
-
10% remaining activity, R102 is part of quinolinic acid binding site, denotes from the other subunit in the canonical dimer
R102Q
-
10% remaining activity, R102 is part of quinolinic acid binding site, denotes from the other subunit in the canonical dimer
R138Q
-
inactive, R138 is part of quinolinic acid binding site
R161A
-
20% remaining activity, R161 is part of quinolinic acid binding site and important for substrate binding
R161Q
-
inactive, R161 is part of quinolinic acid binding site
D235A
-
does not affect ligand binding or catalysis, KD value for quinolinic acid is similar to wild-type, increases KD value for 5-phospho-alpha-D-ribose 1-diphosphate by 2fold
E214A
-
increases KD value for quinolinic acid by 2fold and for 5-phospho-alpha-D-ribose 1-diphosphate by 15fold, causes at least a 4000fold reduction in kcat. Presence of benzene-1,2-dicarboxylic acid results in 3.4fold tightening of 5-phospho-alpha-D-ribose 1-diphosphate binding
E214D
-
KD value for quinolinic acid is similar to wild-type, increases KD value for 5-phospho-alpha-D-ribose 1-diphosphate by 10fold. Presence of benzene-1,2-dicarboxylic acid results in 6fold tightening of 5-phospho-alpha-D-ribose 1-diphosphate binding
E214Q
-
increases KD value for quinolinic acid by 2fold and for 5-phospho-alpha-D-ribose 1-diphosphate by 2fold, has only modest effects on ligand binding and catalysis, wild-type-like pH profile. Presence of benzene-1,2-dicarboxylic acid results in 2fold tightening of 5-phospho-alpha-D-ribose 1-diphosphate binding
K153A
-
inactive enzyme, kcat value decreases by more than 4000fold, is able to bind quinolinic acid with a KD value 2fold higher than that of wild-type
K185A
-
kcat value is reduced by 625fold, and binding affinity of quinolinic acid and 5-phospho-alpha-D-ribose 1-diphosphate to the enzyme decreases. Displays 83fold increase in activity toward the normally inactive quinolinic acid analogue, nicotinic acid. Displays a 300fold higher kcat/Km for nicotinic acid over the natural substrate quinolinic acid
R118A
-
results in 5000fold decrease in kcat value and a decrease in the binding affinity of quinolinic acid and 5-phospho-alpha-D-ribose 1-diphosphate to mutant R152A. Equimolar mixtures of mutant R118A with inactive or virtually inactive mutants produce approximately 50% of the enzymatic activity of wild-type
R152A
-
kcat value is reduced by 33fold, and binding affinity of quinolinic acid and 5-phospho-alpha-D-ribose 1-diphosphate to the enzyme decreases. Displays 116fold increase in activity toward the normally inactive quinolinic acid analogue, nicotinic acid
K284A
-
KD value for quinolinic acid is similar to wild-type, decreases kcat by 30fold and increases Km and KD values for 5-phospho-alpha-D-ribose 1-diphosphate by 80fold and at least 20fold, respectively
additional information
-
mutants can use 6-aminonicotinic acid as substrate, whereas 2,4-pyridinedicarboxylic acid, 2,5-pyridinedicarboxylic acid, 2,6-pyridinedicarboxylic acid, 3,4-pyridinedicarboxylic acid, 3,5-pyridinedicarboxylic acid, nicotinamide and pyridine-4-carboxylic acid are not utilized by the mutant enzymes