Information on EC 6.3.4.21 - nicotinate phosphoribosyltransferase

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

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EC NUMBER
COMMENTARY hide
6.3.4.21
-
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RECOMMENDED NAME
GeneOntology No.
nicotinate phosphoribosyltransferase
-
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O = beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pentosyl group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NAD salvage pathway I
-
-
pyridine nucleotide cycling (plants)
-
-
NAD metabolism
-
-
Nicotinate and nicotinamide metabolism
-
-
Metabolic pathways
-
-
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SYSTEMATIC NAME
IUBMB Comments
5-phospho-alpha-D-ribose 1-diphosphate:nicotinate ligase (ADP, diphosphate-forming)
The enzyme, which is involved in pyridine nucleotide recycling, can form beta-nicotinate D-ribonucleotide and diphosphate from nicotinate and 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) in the absence of ATP. However, when ATP is available the enzyme is phosphorylated resulting in a much lower Km for nicotinate. The phospho-enzyme is hydrolysed during the transferase reaction, regenerating the low affinity form. The presence of ATP shifts the products/substrates equilibrium from 0.67 to 1100 [4].
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CAS REGISTRY NUMBER
COMMENTARY hide
9030-26-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SB19
-
-
Manually annotated by BRENDA team
SB19
-
-
Manually annotated by BRENDA team
beef
-
-
Manually annotated by BRENDA team
BALB/c
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
male Wistar strain
-
-
Manually annotated by BRENDA team
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
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
additional information
?
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
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
additional information
?
-
P39683
molecular evolution mechanism
-
-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
requirement, slight synergism with ATP, more effective than Mg2+ with or without ATP
Fe2+
-
slight stimulation, synergism with ATP
MgATP2-
Mn2+
-
requirement, 7.5 mM, no synergism with ATP, more effective than Mg2+ with or without ATP
NaF
-
activation
phosphate
Zn2+
-
slight stimulation
additional information
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-acetylpyridine
-
-
3-Pyridylcarbinol
-
weak inhibitor
3-Pyridylcarboxaldehyde
-
-
5-Fluoronicotinic acid
-
-
5-phospho-alpha-D-ribose 1-diphosphate
acetyl-CoA
-
at 1 mM 40% inhibition of activity (concentrations of substrates 1 mM)
CoA
-
at 1 mM 70% inhibition of activity (concentrations of substrates 1 mM)
D-fructose 1,6-bisphosphate
-
at 1 mM 36% inhibition of activity (concentrations of substrates 1 mM)
diphosphate
-
-
GDP
-
-
glutaryl-CoA
-
at 1 mM 42% inhibition of activity (concentrations of substrates 1 mM)
glyceraldehyde 3-phosphate
-
at 1 mM 27% inhibition of activity (concentrations of substrates 1 mM)
GMP
-
-
IDP
-
-
IMP
-
-
ITP
-
-
Methyl 3-pyridylcarbinol
-
weak inhibitor
MgADP-
-
non-competitive to Mg-ATP or Mg-diphosphate
Monoiodoacetic acid
-
1 mM and above
N-ethylmaleimide
-
0.1 mM and above
NADH
-
weak inhibitor
NADP+
NADPH
-
weak inhibitor
Nicotinate analogues
-
nicotinate mononucleotide
nucleoside diphosphates
-
e.g. ADP or GDP
PCMB
-
0.01 mM and above
phosphate
-
0.1 M and above
phosphoenolpyruvate
-
at 1 mM 33% inhibition of activity (concentrations of substrates 1 mM)
Pyrazine 2-carboxylic acid
-
-
Pyridazine-4,5-dicarboxylic acid
-
weak, 10% less activity
succinyl-CoA
-
at 1 mM 53% inhibition of activity (concentrations of substrates 1 mM)
tripolyphosphate
-
-
Urea
-
competitive to nicotinate mononucleotide
UTP
-
weak
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
GSH
-
activation
tripolyphosphate
-
activation, can replace ATP
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00022 - 48
5-phospho-alpha-D-ribose 1-diphosphate
0.07 - 0.4
ATP
0.34
GTP
-
-
1.16
ITP
-
good alternative substrate for ATP
0.02
Mg-5-phospho-alpha-D-ribose 1-diphosphate
-
in the coupled reaction, 200fold lower than in the uncoupled reaction
0.0005 - 0.4
nicotinate
0.0127 - 0.0155
nicotinic acid
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.01 - 0.36
5-phospho-alpha-D-ribose 1-diphosphate
0.012 - 0.25
nicotinate
additional information
additional information
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.452 - 9.42
5-phospho-alpha-D-ribose 1-diphosphate
0.271 - 9.16
nicotinate
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.85
CoA
Homo sapiens
-
pH 7.5, 37°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0012
-
-
0.0169
-
+/-0.006 micromol/min/mg, with respect to nicotinic acid, in presence of 1 mM NAD+, 80 ng enzyme, 10-70 microM nicotinamide, 0.3 mM PRPP, 30 min, 37°C, TLC-based quantification
0.0186
-
+/-0.006 micromol/min/mg, with respect to nicotinic acid, 80 ng enzyme, 10-70 microM nicotinamide, 0.3 mM PRPP, 30 min, 37°C, TLC-based quantification
0.0226
-
+/-0.0016 micromol/min/mg, with respect to 5-phospho-alpha-D-ribose 1-diphosphate, in presence of 1 mM NAD+, 20 ng enzyme, 40 microM nicotinamide, 0.14-1 microM PRPP, 15 min, 37°C, TLC-based quantification
0.0236
-
+/-0.0015 micromol/min/mg, with respect to 5-phospho-alpha-D-ribose 1-diphosphate, 20 ng enzyme, 40 microM nicotinamide, 0.14-1 microM PRPP, 15 min, 37°C, TLC-based quantification
0.052
-
-
0.08
-
recombinant enzyme from Escherichia coli strain BA203, pH and temperature not specified in the publication
0.095
-
crude extract
0.7
-
recombinant enzyme from Escherichia coli strain BA002, pH and temperature not specified in the publication
0.74
-
recombinant enzyme from Escherichia coli strain BA204, pH and temperature not specified in the publication
10.54
-
recombinant enzyme from Escherichia coli strain BA014, pH and temperature not specified in the publication
12.24
-
recombinant enzyme from Escherichia coli strain BA207, pH and temperature not specified in the publication
17.31
-
recombinant enzyme from Escherichia coli strain BA206, pH and temperature not specified in the publication
18.74
-
recombinant enzyme from Escherichia coli strain BA016, pH and temperature not specified in the publication
23.72
-
recombinant enzyme from Escherichia coli strain BA207, pH and temperature not specified in the publication
160.5
-
DEAE-Sephadex fraction
421.2
-
Sephacryl fraction
457.3
-
phenyl-Sepharose fraction
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
-
broad, 0.2 M potassium phosphate buffer or 0.05 M Tris-Cl
7.2
-
broad, phosphorolysis
7.3 - 7.4
-
-
7.5 - 8.5
-
broad
additional information
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8
-
about half-maximal activity at pH 5.5 and about 85% of maximal activity at pH 8
5.5 - 10
-
about half-maximal activity at pH 5.5 and 10
additional information
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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exogenously added nicotinic acid (1-10 microM) leads to increase in basal total NAD++ level (revealed by ESI-MS) in a dose-dependent manner and to NaAD+ accumulation which are reduced by siRNA-based knock-down of endogenous NAPRT, knock-down does not affect basal NAD++ levels (503 +/-104 microM) in absence of exogenous nicotinic acid, exogenously added nicotinic acid decreases oxidative cytotoxicity (by 30-50 microM hydrogen peroxide) through elevated NAD+ levels mediated by NAPRT activity as revealed by siRNA-based knock-down of NAPRT
Manually annotated by BRENDA team
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very low protein expression level (mouse polyclonal anti-human NAPRT antibody) and enzymatic activity (TLC)
Manually annotated by BRENDA team
additional information
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Agrobacterium fabrum (strain C58 / ATCC 33970)
Enterococcus faecalis (strain ATCC 700802 / V583)
Enterococcus faecalis (strain ATCC 700802 / V583)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46000
-
x * 46000, SDS-PAGE
52000
-
overexpressed, His-tagged NAPRT in Hep-G2 cells detected in Western blot using mouse polyclonal anti-human NAPRT antibody
54000
-
purified recombinant NAPRT
55000
-
2 * 55000, non-denaturing polyacrylamide gel electrophoresis of purified recombinant NAPRT in active state
59800
-
calculated from the deduced protein sequence
63000
-
2 * 63000, SDS-PAGE
86000
-
gel filtration
87000
-
gel filtration, predicted value of monomer: 59249 Da
110000
-
non-denaturing polyacrylamide gel electrophoresis of purified recombinant NAPRT in active state as controlled by applying gel slice to TLC-based activity assay with 50 microM nicotinic acid + 0.3 mM PRPP, 3 h, 37°C
120000
-
liver, SDS-PAGE
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
2 * 63000, SDS-PAGE
hexamer
6 * 43000, (alphabeta)3, SDS-PAGE and crystal structure analysis
homodimer
-
2 * 55000, non-denaturing polyacrylamide gel electrophoresis of purified recombinant NAPRT in active state
monomer
additional information
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant selenomethionine-labeled enzyme, 10-30 mg/ml, in 10 mM Tris, pH 7.8, 150 mM NaCl, nanodroplet vapour diffusion method, 4°C, reservoir solution contains 6-16% PEG 5000 monomethyl ether, 0.06 M MES, and 0.04 M NaMES, pH 6.0, usage of 15-20% ethylene glycol as cryoprotectant, X-ray structure determination and analysis at 1.75 A resolution
recombinant detagged wild-type and selenomethionine-labeled enzymes, hanging drop vapour diffusion method, room temperature, 0.001 ml protein solution mixed with equal volume of well solution containing 0.2 M ammonium acetate, 0.1 M Tris-HCl, pH 8.5, 45% 2-methyl-2,4-pentanediol, 1 week, crystallization condition screening using sitting drop vapour diffusion at room temperature, iridium complexed, X-ray diffraction structure determination and analysis at 2.8 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
-
stable, 0.2 M potassium phosphate buffer
637752
6.5
-
inactivation below, phosphorolysis
637749
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
-
5 min: 95% loss of activity, MgATP2-, Mg-phosphoribose diphosphate, ATP, GTP or nicotinic acid protect, with 27%, 37%, 52%, 54% or 61% loss of activity, respectively. 10 min: 97% loss of activity, MgATP2-, Mg-5-phospho-alpha-D-ribose 1-diphosphate, ATP, GTP or nicotinic acid protect, with 43%, 52%, 60%, 63% or 69% loss of activity, respectively
additional information
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
50% activity recovered at salt concentrations of 50 mM, inactivation with salt concentrations below 20 mM, 20-35% glycerol partially protects
-
dialysis against 50 mM Tris-HCl buffer at pH 7.3 in the absence of potassium phosphate, significant loss of activity
first order activity loss in the presence of 1/200 trypsin/enzyme
-
glycerol, 20-35%, stabilizes during dialysis against low buffer concentrations, more highly purified preparations require higher glycerol concentrations
-
heat, unstable to, substrates protect
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rapidly inactivates the ATPase and nicotinic acid mononucleotide synthesis activities with cleavages at Arg-384 and Lys-374, ATP and 5-phospho-alpha-D-ribose 1-diphosphate increase the t1/2 for inactivation 8fold and 4fold, respectively, and 32fold, together
-
sensitive to trypsin, partial proteolysis, 4fold increase in t1/2 for inactivation in the presence of 1 mM MgATP2-
-
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-12°C, partially purified enzyme preparation, t1/2: at least 3 months
-
-25°C, at least 1 month
-
-70°C, t1/2: 3 months
-
-70ºC, no significantly decreased activity for up to 3 years, no significantly decreased after 8 years
-
4ºC, as 70% saturated ammonium sulfate suspensions in 200 mM phosphate buffer, pH 8, 10% glycerol and 5 mM DTT
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; nickel chelate His-bind resin after bacterial expression
-
ammonium hydroxide precipitation, chromatography on DEAE-Sephadex, Sephacryl and phenyl-Sepharose
-
ammonium sulfate fractionation, chromatography on DEAE-cellulose, chromatography on Sephadex, affinity column chromatography and hydroxylapatite column chromatography, 30000fold purified
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bacteria disruption by alumina grinding, protamine sulfate precipitation and chromatography on DEAE-cellulose
-
bacterial suspension disruption and centrifugation, chromatography on Q-Sepharose and phenyl-Sepharose
-
bacterial suspension disruption and centrifugation, chromatography on Sephadex-G, DEAE-Toyopearl, Cibacron Blue and Sephacryl gel filtration
-
extraction, ammonium sulfate fractionation and chromatography on DEAE-cellulose
-
extraction, ammonium sulfate fractionation, chromatography on DEAE-cellulose and hydroxylapatite, 1000fold purified
-
extraction, fractionation chromatography on phosphocellulose, hydroxyapatite, blue Sephadex and DEAE-cellulose
-
heparinised blood lysates
-
homogenates
-
immobilized metal ion affinity chromatography (Ni2+)
-
partial, extraction, ammonium sulfate fractionation and chromatography on DEAE-cellulose, 60fold purified
-
recombinant N-terminally tagged selenomethionine-labeled enzyme variant from Escherichia coli strain DL41
recombinant tagged wild-type and selenomethionine-labeled fusion enzymes from Escherichia coli to homogeneity by high-trap chelating nickel affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA fragment (as probe for Northern blot analysis) reverse transcribed from tissue total RNA using sense primer 5’-GTG AGG TGA ATG TCA TTG GC-3’ and antisense primer 5’-ACA GTG CGA CCG GAT ACA CT-3’
expressed in Escherichia coli NZN111
-
expressed in Escherichia coli strains BA002, BA014, BA015, and BA016
-
expressed in Escherichia coli strains BA002, BA014, BA026, and BA207
-
expressed in Escherichia coli strains BA203, BA204, BA206, and BA209
-
expression in Escherichia coli
-
expression in Escherichia coli as His6-tagged protein; in pET22b and pET15b for expression in Escherichia coli BL21 (DE3) as fusion protein with C-terminal or N-terminal hexa-His-tag, respectively, in pcDNA3His(6) for expression in eukaryotic cell culture, specific siRNAs against nt829-1634 and nt445-935 for transfection into HEK-293 cells
-
expression of N-terminally tagged enzyme as selenomethionine-labeled variant in Escherichia coli strain DL41
expression of pncB gene on a multicopy pUC19 plasmid in Salmonella typhimurium
-
expression of the enzyme as His6-tagged-maltose-binding fusion protein containing a tobacco etch virus protease cleavage site in Escherichia coli strain BL21(DE3) for the wild-type, and in strain B834(DE3) for a selenomethionine-labeled variant
His-tagged protein expressed in Escherichia coli BL21
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D19A
-
activity completely abolished
D288A
-
about 10% of wild type activity
G169A
-
about 40% of wild type activity
G209A
-
about 20% of wild type activity
G379A
-
activity completely abolished
H213A
-
about 50% of wild type activity
N357A
-
about 80% of wild type activity
R318A
-
activity strictly dependent on the presence of ATP
S381A
-
about 40% of wild type activity
T380A
-
about 50% of wild type activity
Y21A
-
activity strictly dependent on the presence of ATP
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LINKS TO OTHER DATABASES (specific for EC-Number 6.3.4.21)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)