Information on EC 6.3.4.21 - nicotinate phosphoribosyltransferase

LOC_Os03g62110

gene name

NA phosphoribosyltransferase

NAPRT

NaPRT1

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NAPRTase

niacin ribonucleotidase

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nicotinate phosphoribosyltransferase

nicotinate phosphoribosyltransferases

nicotinate-nucleotide:pyrophosphate phospho-alpha-D-ribosyltransferase

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nicotinic acid mononucleotide glycohydrolase

-

nicotinic acid mononucleotide pyrophosphorylase

nicotinic acid phosphoribosyltransferase

8 entries

NPRTase

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637754, 637755

pncB

additional information

-

enzyme is a member of the phosphoribosyltransferase superfamily or PRTase superfamily

nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O = beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate

pentosyl group transfer

Nicotinate and nicotinamide metabolism

BRENDA

NAD metabolism

KEGG

MetaCyc

NAD salvage pathway I (PNC VI cycle), NAD salvage pathway V (PNC V cycle), pyridine nucleotide cycling (plants)

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].

9030-26-6

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nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O

beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate

40 entries

additional information

?

-

14 entries

nicotinate + 5-phospho-alpha-D-ribose 1-diphosphate + ATP + H2O

beta-nicotinate D-ribonucleotide + diphosphate + ADP + phosphate

16 entries

additional information

?

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P39683

molecular evolution mechanism

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ATP

CTP

GTP

ITP

TTP

UTP

Co2+

-

requirement, slight synergism with ATP, more effective than Mg2+ with or without ATP

Fe2+

-

slight stimulation, synergism with ATP

Mg2+

MgATP2-

Mn2+

-

requirement, 7.5 mM, no synergism with ATP, more effective than Mg2+ with or without ATP

NaF

-

activation

637748

phosphate

Zn2+

-

slight stimulation

additional information

2-hydroxynicotinic acid

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3-acetylpyridine

-

3-Pyridylcarbinol

-

weak inhibitor

3-Pyridylcarboxaldehyde

-

5-Fluoronicotinic acid

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5-phospho-alpha-D-ribose 1-diphosphate

acetyl-CoA

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at 1 mM 40% inhibition of activity (concentrations of substrates 1 mM)

ATP

CoA

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at 1 mM 70% inhibition of activity (concentrations of substrates 1 mM)

D-fructose 1,6-bisphosphate

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at 1 mM 36% inhibition of activity (concentrations of substrates 1 mM)

diphosphate

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GDP

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glutaryl-CoA

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at 1 mM 42% inhibition of activity (concentrations of substrates 1 mM)

glyceraldehyde 3-phosphate

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at 1 mM 27% inhibition of activity (concentrations of substrates 1 mM)

GMP

-

IDP

-

IMP

-

ITP

-

KCN

-

Methyl 3-pyridylcarbinol

-

weak inhibitor

Mg2+

MgADP-

-

non-competitive to Mg-ATP or Mg-diphosphate

637756

Monoiodoacetic acid

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1 mM and above

N-ethylmaleimide

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0.1 mM and above

NAD+

NADH

-

weak inhibitor

NADP+

NADPH

-

weak inhibitor

Nicotinate analogues

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nicotinate mononucleotide

nucleoside diphosphates

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e.g. ADP or GDP

PCMB

-

0.01 mM and above

phosphate

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0.1 M and above

phosphoenolpyruvate

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at 1 mM 33% inhibition of activity (concentrations of substrates 1 mM)

Pyrazine 2-carboxylic acid

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Pyridazine-4,5-dicarboxylic acid

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weak, 10% less activity

succinyl-CoA

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at 1 mM 53% inhibition of activity (concentrations of substrates 1 mM)

tripolyphosphate

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Urea

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competitive to nicotinate mononucleotide

UTP

-

weak

additional information

-

ATP

GSH

-

activation

637748

tripolyphosphate

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activation, can replace ATP

additional information

3 entries

-

0.00022 - 48

5-phospho-alpha-D-ribose 1-diphosphate

16 entries

0.07 - 0.4

ATP

0.34

GTP

-

Salmonella enterica subsp. enterica serovar Typhimurium

1.16

ITP

-

good alternative substrate for ATP

637758

0.02

Mg-5-phospho-alpha-D-ribose 1-diphosphate

Salmonella enterica subsp. enterica serovar Typhimurium

-

in the coupled reaction, 200fold lower than in the uncoupled reaction

637758

0.0005 - 0.4

nicotinate

15 entries

0.0127 - 0.0155

nicotinic acid

0.01 - 0.36

5-phospho-alpha-D-ribose 1-diphosphate

0.012 - 0.25

nicotinate

additional information

-

-

0.452 - 9.42

5-phospho-alpha-D-ribose 1-diphosphate

0.271 - 9.16

nicotinate

0.85

CoA

Homo sapiens

-

pH 7.5, 37°C

0.0012

-

0.0169

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+/-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

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+/-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

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+/-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

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+/-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

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0.08

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recombinant enzyme from Escherichia coli strain BA203, pH and temperature not specified in the publication

0.095

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crude extract

0.7

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recombinant enzyme from Escherichia coli strain BA002, pH and temperature not specified in the publication

726780, 726892

0.74

-

recombinant enzyme from Escherichia coli strain BA204, pH and temperature not specified in the publication

1.01

2.3

-

Salmonella enterica subsp. enterica serovar Typhimurium

3

-

10.54

-

recombinant enzyme from Escherichia coli strain BA014, pH and temperature not specified in the publication

726780, 726892

12.24

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recombinant enzyme from Escherichia coli strain BA207, pH and temperature not specified in the publication

726892

17.31

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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

726780

23.72

-

recombinant enzyme from Escherichia coli strain BA207, pH and temperature not specified in the publication

160.5

-

DEAE-Sephadex fraction

421.2

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Sephacryl fraction

457.3

-

phenyl-Sepharose fraction

6.5 - 8

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broad, 0.2 M potassium phosphate buffer or 0.05 M Tris-Cl

7.2

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broad, phosphorolysis

7.3 - 7.4

-

7.5 - 8.5

-

broad

8.5

additional information

5.5 - 8

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about half-maximal activity at pH 5.5 and about 85% of maximal activity at pH 8

5.5 - 10

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about half-maximal activity at pH 5.5 and 10

6.5 - 9

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Mycobacterium tuberculosis

additional information

37

SB19

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Bacillus subtilis SB19

SB19

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beef

-

-

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BALB/c

UniProt

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637763

P9WJW5

UniProt

Mycobacterium tuberculosis H37Rv

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637763

P9WJW5

UniProt

Rattus norvegicus male Wistar

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UniProt

-

-

male Wistar strain

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Salmonella enterica subsp. enterica serovar Typhimurium

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Uniprot

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weak expression

-

-

-

weak expression

745944

Caki-2 cell

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weak expression

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cell suspension culture

-

-

-

Phoenix cell

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-

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very low protein expression level (mouse polyclonal anti-human NAPRT antibody) and enzymatic activity (TLC)

-

-

-

-

-

additional information

physiological function

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both NAPRT and nicotinamide phosphoribosyltransferase NAMPT increase intracellular NAD+ levels. NAPRT silencing reduces energy status, protein synthesis, and cell size in ovarian and pancreatic cancer cells. NAPRT silencing sensitizes cells to NAMPT inhibitors both in vitro and in vivo

744621

1ybe, download, 3D-view

SCOPe (1ybe)

CATH (1ybe)

Q8UIS9

Agrobacterium fabrum (strain C58 / ATCC 33970)

4yub, download, 3D-view

1yir, download, 3D-view

SCOPe (1yir)

CATH (1yir)

Q9HW26

Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

1vlp, download, 3D-view

SCOPe (1vlp)

CATH (1vlp)

Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Salmonella enterica subsp. enterica serovar Typhimurium

Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)

4hl7, download, 3D-view

SCOPe (4hl7)

CATH (4hl7)

Q9KN67

Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)

3os4, download, 3D-view

43000

45000

45500

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mutant enzyme, SDS-PAGE

Salmonella enterica subsp. enterica serovar Typhimurium

45529

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1 * 45529, gel filtration

Salmonella enterica subsp. enterica serovar Typhimurium

45530

P22253

predicted from DNA sequence

637763

45540

Salmonella enterica subsp. enterica serovar Typhimurium

-

SDS-PAGE

Salmonella enterica subsp. enterica serovar Typhimurium

45550

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mutant enzyme, SDS-PAGE

46000

-

x * 46000, SDS-PAGE

722141

51000

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

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2 * 55000, non-denaturing polyacrylamide gel electrophoresis of purified recombinant NAPRT in active state

59800

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calculated from the deduced protein sequence

63000

-

2 * 63000, SDS-PAGE

86000

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gel filtration

87000

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gel filtration, predicted value of monomer: 59249 Da

110000

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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

?

dimer

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2 * 63000, SDS-PAGE

hexamer

6 * 43000, (alphabeta)3, SDS-PAGE and crystal structure analysis

homodimer

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2 * 55000, non-denaturing polyacrylamide gel electrophoresis of purified recombinant NAPRT in active state

monomer

additional information

by molecular replacement at a resolution of 2.9 A, in its ligand-free form. The enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. NaPRTase acks a tunnel that, in nicotinamide phosphoribosyltransferase EC 2.4.2.12, represents the binding site of inhibitor FK866

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744914

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

D19A

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activity completely abolished

D288A

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about 10% of wild type activity

G169A

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about 40% of wild type activity

G209A

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about 20% of wild type activity

G379A

-

activity completely abolished

H213A

N357A

-

about 80% of wild type activity

R318A

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activity strictly dependent on the presence of ATP

S381A

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about 40% of wild type activity

T380A

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about 50% of wild type activity

Y21A

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activity strictly dependent on the presence of ATP

P21L

mutation identified in a strain with early leaf sensescence, mutation causes the expression of the gene at the transcriptional level to decline by 40% in fully expanded leaves. Leaf nicotinate and nicotinamide contents are elevated, while NAD levels are reduced. Leaf tissue of P21L exhibits significant DNA fragmentation and H2O2 accumulation, along with up-regulation of genes associated with senescence. The mutant also shows reduced expression of SIR2-like genes SRT1 and SRT2 and increased acetylation of histone H3K9

5 - 10

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stable, 0.2 M potassium phosphate buffer

637752

6.5

Salmonella enterica subsp. enterica serovar Typhimurium

-

inactivation below, phosphorolysis

637749

8.3

P22253

native enzyme, unstable

637763

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

50% activity recovered at salt concentrations of 50 mM, inactivation with salt concentrations below 20 mM, 20-35% glycerol partially protects

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dialysis against 50 mM Tris-HCl buffer at pH 7.3 in the absence of potassium phosphate, significant loss of activity

Salmonella enterica subsp. enterica serovar Typhimurium

first order activity loss in the presence of 1/200 trypsin/enzyme

-

637759, 637760

glycerol, 20-35%, stabilizes during dialysis against low buffer concentrations, more highly purified preparations require higher glycerol concentrations

-

heat, unstable to, substrates protect

-

Salmonella enterica subsp. enterica serovar Typhimurium

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

-

637760

sensitive to trypsin, partial proteolysis, 4fold increase in t1/2 for inactivation in the presence of 1 mM MgATP2-

Salmonella enterica subsp. enterica serovar Typhimurium

-

-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

Salmonella enterica subsp. enterica serovar Typhimurium

-

637762

4ºC, as 70% saturated ammonium sulfate suspensions in 200 mM phosphate buffer, pH 8, 10% glycerol and 5 mM DTT

-

637759, 637760, 637761

t1/2: at least 2 months

Salmonella enterica subsp. enterica serovar Typhimurium

-

-

-

637756

; nickel chelate His-bind resin after bacterial expression

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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

-

bacteria disruption by alumina grinding, protamine sulfate precipitation and chromatography on DEAE-cellulose

Salmonella enterica subsp. enterica serovar Typhimurium

-

637748

bacterial suspension disruption and centrifugation, chromatography on Q-Sepharose and phenyl-Sepharose

-

637760

bacterial suspension disruption and centrifugation, chromatography on Sephadex-G, DEAE-Toyopearl, Cibacron Blue and Sephacryl gel filtration

Salmonella enterica subsp. enterica serovar Typhimurium

-

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

-

637753

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

-

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’

Mus musculus

Q8CC86

expressed in Escherichia coli NZN111

-

722141

expressed in Escherichia coli strains BA002, BA014, BA015, and BA016

-

726780

expressed in Escherichia coli strains BA002, BA014, BA026, and BA207

-

726892

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

Salmonella enterica subsp. enterica serovar Typhimurium

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

-

presence of more than one NAPRT transcript in most tissues, all with identical 3' end sequence. Some NAPRT transcripts appear to be tissuespecific. Brain shows the highest number of alternative transcripts. Several modulators of the NAPRT gene expression are involved, i.e. mutations in transcription factor binding sites, promoter methylation and alternative splicing

-

745944

medicine