Information on EC 6.3.1.5 - NAD+ synthase

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

EC NUMBER
COMMENTARY
6.3.1.5
-
RECOMMENDED NAME
GeneOntology No.
NAD+ synthase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ATP + deamido-NAD+ + NH3 = AMP + diphosphate + NAD+
show the reaction diagram
flexible active site loops of residues 82-7 and 204-225, stabilization of the loop and active site structure, mechanism
-
ATP + deamido-NAD+ + NH3 = AMP + diphosphate + NAD+
show the reaction diagram
mechanism of two-step procedure, reaction intermediate
P08164
ATP + deamido-NAD+ + NH3 = AMP + diphosphate + NAD+
show the reaction diagram
reaction mechanism, determination of deamido-NAD+-binding site, Asp173 is the key residue in both deprotonation of the primarily bound ammonium ion, and stabilization of the tetrahedral transition-state intermediate
P08164
ATP + deamido-NAD+ + NH3 = AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Acid amide hydrolysis
-
-
carboxylic
-
amide bond formation
E07950, -
-
amide bond formation
-
-
amide bond formation
-
-
amide bond formation
-
-
amide bond formation
-
-
amide bond formation
Geobacillus stearothermophilus H-804, Streptococcus sobrinus 6715
-
-
-
amide group transfer
E07950, -
-
amide group transfer
-
-
amide group transfer
-
-
amide group transfer
-
-
amide group transfer
-
-
amide group transfer
Geobacillus stearothermophilus H-804, Streptococcus sobrinus 6715
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Metabolic pathways
-
NAD biosynthesis I (from aspartate)
-
Nicotinate and nicotinamide metabolism
-
SYSTEMATIC NAME
IUBMB Comments
Deamido-NAD+:ammonia ligase (AMP-forming)
L-Glutamine also acts, more slowly, as amido-donor [cf. EC 6.3.5.1].
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
ammonia-dependent NADS
-
-
ammonia-dependent nicotinamide adenine dinucleotide synthetase
-
-
ammonia-specific NAD synthase
-
-
ammonia-specific NAD synthetase
E07950
-
ammonia-specific NAD synthetase
Geobacillus stearothermophilus H-804
E07950
-
-
Diphosphopyridine nucleotide synthetase
-
-
-
-
NAD synthase
-
-
NAD synthase
E07950
-
NAD synthase
Geobacillus stearothermophilus H-804
E07950
-
-
NAD synthase
-
-
NAD synthase
-
-
-
NAD synthetase
-
-
-
-
NAD synthetase
-
-
NAD synthetase
E07950
-
NAD synthetase
Geobacillus stearothermophilus H-804
E07950
-
-
NAD synthetase
-
-
NAD synthetase
-
-
NAD synthetase
Staphylococcus aureus 8325-4
-
-
-
NAD synthetase
-
-
NAD synthetase
-
-
-
NAD+ synthetase
-
-
-
-
NAD+ synthetase
-
-
NadE
Staphylococcus aureus 8325-4
-
-
-
NH3-dependent NAD+ synthetase
-
-
NH3-dependent NAD+ synthetase
-
-
Nicotinamide adenine dinucleotide synthetase
-
-
-
-
Nicotinamide adenine dinucleotide synthetase
-
-
Nicotinamide adenine dinucleotide synthetase
-
-
Nicotinamide adenine dinucleotide synthetase
E07950
-
Nicotinamide adenine dinucleotide synthetase
Geobacillus stearothermophilus H-804
E07950
-
-
Nicotinamide adenine dinucleotide synthetase
-
-
Nicotinamide adenine dinucleotide synthetase
-
-
Nicotinamide adenine dinucleotide synthetase
-
-
-
Qns1
-
couples a glutamine amidotransferase domain to a second active site that requires ammonia as a reactant
Synthetase, nicotinamide adenine dinucleotide
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9032-69-3
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
purified recombinant enzyme
Uniprot
Manually annotated by BRENDA team
wild-type and mutant enzyme
-
-
Manually annotated by BRENDA team
strain B
-
-
Manually annotated by BRENDA team
commercial preparation
-
-
Manually annotated by BRENDA team
H-804
E07950
GenBank
Manually annotated by BRENDA team
Geobacillus stearothermophilus H-804
H-804
E07950
GenBank
Manually annotated by BRENDA team
strain LT2, wild-type and mutant strains which are defective in NAD synthetase
-
-
Manually annotated by BRENDA team
strain 8325-4
-
-
Manually annotated by BRENDA team
Staphylococcus aureus 8325-4
strain 8325-4
-
-
Manually annotated by BRENDA team
strain 6715
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-, Q2A4B6
existence of an alternative route of NAD synthesis where the amidation of NaMN to nicotinamide mononucleotide occurs before the adenylylation reaction, which converts the intermediate to the NAD cofactor. The first step is catalyzed by Francisella tularensis NadE
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-fluoro-ATP + deamido-NAD+ + NH3
2-fluoro-AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
?
ATP + deamido-NAD+ + Asp
AMP + diphosphate + NAD+ + oxaloacetate
show the reaction diagram
-
slightly active as nitrogen donor
-
-
-
ATP + deamido-NAD+ + Glu
AMP + diphosphate + NAD+ + 2-oxoglutarate
show the reaction diagram
-
slightly active as nitrogen donor
-
-
-
ATP + deamido-NAD+ + glutamine
AMP + diphosphate + glutamate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + glutamine
AMP + diphosphate + NAD+ + glutamate
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + Glu
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + Glu
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + Glu
show the reaction diagram
-
ineffective as amide donor
-
-
-
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
P08164
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
ir
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-, Q2A4B6
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-, Q81RP3
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
E07950, -
-
-
ir
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
NH3 is the preferred nitrogen donor
-
-
-
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
NH3 is the preferred nitrogen donor
-
-
-
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
dependent on NH3
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
P08164
NAD-adenylate intermediate, two step reaction
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
E07950, -
specific for NH3, no activity with L-glutamine or other L-amino acids, no activity with urea, uric acid, or creatinine
-
ir
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
P08164
NAD-adenylate intermediate, key two step reaction in NAD+ biosynthesis
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
Geobacillus stearothermophilus H-804
E07950
-, specific for NH3, no activity with L-glutamine or other L-amino acids, no activity with urea, uric acid, or creatinine
-
ir
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
Staphylococcus aureus 8325-4
-
-
-
-
?
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
last step in the metabolic pathway for the biosynthesis of NAD
-
-
-
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
last step in the metabolic pathway for the biosynthesis of NAD
-
-
-
ATP + deamido-NAD+ + NH4+
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
?
ATP + deamido-NAD+ + NH4+
AMP + diphosphate + NAD+ + H+
show the reaction diagram
-
-
-
?
ATP + deamido-NMN + NH3
AMP + diphosphate + NMN
show the reaction diagram
-, Q2A4B6
existence of an alternative route of NAD synthesis where the amidation of NaMN to nicotinamide mononucleotide occurs before the adenylylation reaction, which converts the intermediate to the NAD cofactor. The first step is catalyzed by Francisella tularensis NadE
-
-
?
dATP + deamido-NAD+ + NH3
dAMP + diphosphate + NAD+
show the reaction diagram
-
20% of the activity relative to ATP
-
-
-
additional information
?
-
-
2 possible substrates, NH3 or glutamine, to perform the final step in the Preiss-Handler pathway for NAD+ biosynthesis
-
?
additional information
?
-
-
enzyme induces lymphocyte polyclonal activation, it stimulates murine B cells after in vitro treatment of spleen cell cultures
-
?
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
ATP + deamido-NAD+ + glutamine
AMP + diphosphate + glutamate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
P08164
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
E07950, -
-
-
ir
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
P08164
NAD-adenylate intermediate, key two step reaction in NAD+ biosynthesis
-
?
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
-
-
-
-
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
last step in the metabolic pathway for the biosynthesis of NAD
-
-
-
ATP + deamido-NAD+ + NH3
?
show the reaction diagram
-
last step in the metabolic pathway for the biosynthesis of NAD
-
-
-
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
Geobacillus stearothermophilus H-804
E07950
-
-
ir
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH4+
AMP + diphosphate + NAD+ + H+
show the reaction diagram
-
-
-
?
additional information
?
-
-
2 possible substrates, NH3 or glutamine, to perform the final step in the Preiss-Handler pathway for NAD+ biosynthesis
-
?
additional information
?
-
-
enzyme induces lymphocyte polyclonal activation, it stimulates murine B cells after in vitro treatment of spleen cell cultures
-
?
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
K+
P08164
required, 2 binding sites near the bound diphosphate at the ATP-binding site, can be substituted by Tl+
Mg2+
-
required, binding structure and mechanism, 2 binding sites per molecule of enzyme
Mg2+
P08164
required
Mg2+
E07950, -
required, optimal at 3 mM
Mg2+
-
required
Mg2+
-
required at 1.5 mM
Mg2+
P08164
2 Mg2+ ligands per molecule of enzyme
Mg2+
-, Q81RP3
required
Mg2+
-
can satisfy the metal ion requirement, Mn2+ is more effective than Mg2+ at low concentrations, at high concentrations Mn2+ markedly inhibits, but Mg2+ does not
Mn2+
-
can satisfy the metal ion requirement, Mn2+ is more effective than Mg2+ at low concentrations, at high concentrations Mn2+ markedly inhibits, but Mg2+ does not
Tl+
P08164
can substitute for K+
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1-(4-benzyloxyphenoxy)-8-(2-guanidino-2-phenyl-1-ethyloxy)-octane
-
-
1-(4-benzyloxyphenoxy)-8-(2-guanidino-3-phenyl-1-propyloxy)-octane
-
-
1-(4-benzyloxyphenoxy)-8-(2-N,N,N-trimethylammonium-2-phenyl-1-ethyloxy)octane
-
-
1-(4-benzyloxyphenoxy)-8-(2-N,N,N-trimethylammonium-3-phenyl-1-propyloxy)octane
-
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-(1H-indol-3-yl)propionate
-
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-(4-hydroxyphenyl)propionate
-
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-phenylpropionate
-
-
adenosine
-
-
Ciprofloxacin
-
-
clotrimazole
-
-
Mg2+
-
slightly inhibiting above 5 mM
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-(N,N,N-trimethylammonium)-2-phenylacetamide
-
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-(N,N,N-trimethylammonium)-3-phenylpropionamide
-
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-guanidino-2-phenylacetamide
-
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-guanidino-3-phenylpropionamide
-
-
Psicofuranine
-
i.e. 9-D-psicofuranosyl-6-aminopurine, each substrate provides some protection
Thrombin
-
enzyme is highly susceptible too proteolytic cleavage
-
Mn2+
-
can satisfy the metal ion requirement at low concentrations, at high concentrations Mn2+ markedly inhibits
additional information
-
clotrimazole does not inhibit in the presence of 0.01% Triton X-100
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00079
-
ATP
-
pH 8.5, 37C, recombinant truncated enzyme
0.0013
-
ATP
-
pH 8.5, 37C, recombinant full length enzyme
0.2
-
ATP
-
in 60 mM Tris-HCl pH 8.0, 10 mM NH4Cl, 10 mM MgCl2, 10 mM KCl
0.2
-
ATP
-
-
0.289
-
ATP
-, Q81RP3
in 60 mM EPPS pH 8.5 with 20 mM ammonium chloride, 20 mM potassium chloride, and 10 mM magnesium chloride, at 37C
0.00052
-
deamido-NAD+
-
pH 8.5, 37C, recombinant truncated enzyme
0.0029
-
deamido-NAD+
-
pH 8.5, 37C, recombinant full length enzyme
0.03
-
deamido-NAD+
-
in 60 mM Tris-HCl pH 8.0, 10 mM NH4Cl, 10 mM MgCl2, 10 mM KCl
0.11
-
deamido-NAD+
-
-
0.152
-
deamido-NAD+
-, Q81RP3
in 60 mM EPPS pH 8.5 with 20 mM ammonium chloride, 20 mM potassium chloride, and 10 mM magnesium chloride, at 37C
5.82
-
deamido-NAD+
-, Q2A4B6
-
0.2
-
deamido-NMN
-, Q2A4B6
-
0.0011
-
glutamine
-
pH 8.5, 37C, recombinant full length enzyme
0.0016
-
glutamine
-
pH 8.5, 37C, recombinant truncated enzyme
16
-
L-Gln
-
-
0.065
-
NH3
-
-
0.91
-
NH3
E07950, -
pH 8.5, 37C
0.025
-
NH4+
-
pH 8.5, 37C, recombinant truncated enzyme
0.027
-
NH4+
-
pH 8.5, 37C, recombinant full length enzyme
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.25
-
deamido-NAD+
-, Q2A4B6
-
0.5
-
deamido-NMN
-, Q2A4B6
-
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
43
-
deamido-NAD+
-, Q2A4B6
-
9516
2500
-
deamido-NMN
-, Q2A4B6
-
293380
additional information
-
additional information
-
the Vmax/KM-value for NADsyn2 with NH3 is 2370fold higher than the Vmax/Km-value for glutamine
0
IC50 VALUE [mM]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.011
-
1-(4-benzyloxyphenoxy)-8-(2-guanidino-2-phenyl-1-ethyloxy)-octane
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.011
-
1-(4-benzyloxyphenoxy)-8-(2-guanidino-3-phenyl-1-propyloxy)-octane
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.038
-
1-(4-benzyloxyphenoxy)-8-(2-N,N,N-trimethylammonium-2-phenyl-1-ethyloxy)octane
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.021
-
1-(4-benzyloxyphenoxy)-8-(2-N,N,N-trimethylammonium-3-phenyl-1-propyloxy)octane
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.019
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-(1H-indol-3-yl)propionate
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.053
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-(4-hydroxyphenyl)propionate
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.037
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-phenylpropionate
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5, in the presence of 0.01% Triton X-100
0.22
-
8-(4-benzyloxyphenoxy)-1-octyl 2-(N,N,N-trimethylammonium)-3-phenylpropionate
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.045
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-(N,N,N-trimethylammonium)-2-phenylacetamide
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.04
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-(N,N,N-trimethylammonium)-3-phenylpropionamide
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.019
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-guanidino-2-phenylacetamide
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
0.012
-
N-[8-(4-benzyloxyphenoxy)-1-octyl]-2-guanidino-3-phenylpropionamide
-
in 58.5 mM HEPPS buffer, 18.5 mM NH4Cl, 19.5 mM KCl, 9.75 mM MgCl2, 1% (v/v) EtOH, at pH 8.5
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.0003
-
-
mutant enzyme R112L, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.0011
-
-
mutant enzyme D593A/F622A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.0027
-
-
mutant enzyme L604A/L519A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.0033
-
-
mutant enzyme Y532A/M621A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.0043
-
-
mutant enzyme L604N, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.008
-
-
mutant enzyme F622A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.009
-
-
partially purified recombinant His-tagged enzyme expressed in insect cells, substrate NH4+
0.0143
-
-
mutant enzyme I111A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.015
-
-
mutant enzyme R112S, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.033
-
-
purified recombinant HIs-tagged enzyme expressed in Escherichia coli, substrate glutamine
0.0485
-
-
mutant enzyme Y601A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.1
-
-
purified recombinant His-tagged enzyme expressed in Escherichia coli, substrate NH4+
0.152
-
-
mutant enzyme D593A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.183
-
-
mutant enzyme L604A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.25
-
-
mutant enzyme M621A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.49
-
-
purified recombinant His-tagged enzyme
0.503
-
-
-
0.631
-
-
mutant enzyme E177A, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
0.67
-
-
purified recombinant His-tagged truncated enzyme expressed in Escherichia coli, substrate NH4+
0.741
-
-
wild type enzyme, at 37C, with 2 mM ATP, 5 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 56 mM KCl, and 0.2 mg/ml bovine serum albumin
1.5
-
-
recombinant enzyme, at 37C
1.59
-
E07950, -
purified native enzyme
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
-
-, Q81RP3
-
8.4
8.6
-
-
8.5
-
-
assay at
8.5
-
-
with substrate NH4+
8.8
-
-
with substrate glutamine
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8.2
8.7
-, Q81RP3
-
additional information
-
-
active in a broad pH range
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.3
-
E07950, -
isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Burkholderia pseudomallei (strain 1710b)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain NCTC 11168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. holarctica (strain LVS)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Vibrio cholerae serotype O1 (strain MJ-1236)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
50000
-
E07950, -
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 38000, recombinant His-tagged enzyme, SDS-PAGE
?
-
x * 35000, recombinant enzyme, SDS-PAGE
?
-
x * 38000, recombinant His-tagged enzyme, SDS-PAGE
-
dimer
E07950, -
2 * 25000, SDS-PAGE
dimer
P08164
homodimer, crystal structure determination
dimer
-
2 * 30240
dimer
Geobacillus stearothermophilus H-804
-
2 * 25000, SDS-PAGE
-
additional information
-
multimeric enzyme of at least two subunits
additional information
-
x * 60480, calculation from nucleotide sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
hanging drop vapour diffusion method using 8-12% PEG 8000, 0.505 M ammonium sulfate, 6% glycerol, 100 mM magnesium chloride, 0.05% n-octyl-beta-D-glucopyranoside, 100 mM HEPES, pH 7
-, Q81RP3
enzyme in complex with ATP, ATP-Tl+, ATP-Mn2+, or NAD-adenylate, hanging-drop vapour diffusion method, 15 mg/ml protein, 5 mM ATP, 20 mM NAD+ in 0.1 M sodium acetate, pH 5.2, 22% PEG 400, and 50 mM MgCl2, thallium acetate, or MnCl2 20C, 24 h before data collection the crystale are soaked in a cryoprotectant buffer, X-ray diffraction structure determination at 1.3 A resolution, structure analysis and modeling
P08164
free enzyme form at 2.6 A resolution and in a complex with ATP at 2.0 A resolution
-
purified recombinant enzyme complexed with substrates, vapour diffusion method at 5.2 under earth gravity at pH 5.2, and microseeding under space microgravity, the latter in protein solution containing 15 mg/ml protein, 2.5 mM ATP, 5 mM deamido-NAD+, 50 mM Tris, pH 8.5, 50 mM MgCl2, 25% PEG 400 v/v, 9 days, X-ray diffractio structure determination at 1.0 A resolution and analysis of the crystal structures at pH 5.2 and pH 8.5
P08164
purified recombinant enzyme, in complex with 4 different substrates: complex I is formed by enzyme and deamido-NAD, complex II is formed by enzyme and ATP, complex III is formed by enzyme, deamido-NAD and ATP, complex is formed by enzyme and substrate analogue alpha,beta-methylene-adenosine triphosphate, crystallization from protein solution: 15 mg/ml protein, 20 mM sodium acetate, pH 5.2, 50 mM MgCl2, 2.5 mM 2-mercaptoethanol, plus equal volume of 0.1 M sodium acetate, pH 5.2, 21-23% PEG 400, 50 mM MgCl2, at room temperature, addition of substrates at different concentrations, formation of complex IV at different pH-values, in the reservoir solution: 50 mM HEPES, pH 7.5, 0.1 M MgCl2, 20-26% PEG 400, 2 mM AMP-CPP, X-ray diffraction structure determination at resolution 1.9-2.3, structure analysis
-
28 mg/ml purified recombinant enzyme, pure or in complex with substrates NAD+ and ATP, hanging-drop vapour diffusion method, 22C room temperature, protein in 0.01 M HEPES, pH 7.5, 5 mM MgCl2, 3 mM DTT, precipitant solution: 0.1 M HEPES, pH 7.5, 1.5 M lithium sulfate, X-ray diffraction structure determination at 2.0 A resolution, compex structure determination via molecular replacement method
-
crystal structures of the enzyme alone and in complex with natural substrates and with the reaction product NAD+
-
to 1.9 A resolution
-, Q2A4B6
crystal structures of apo- and complex forms with deamido-NAD+ and ATP to a resolution of 2.3 and 1.7 respectively. Hanging drop vapor diffusion method. Crystals of SeMet-containing NAD+ synthase belong to space group C2 with unit cell dimensions of a = 92.3, b = 47.5, c = 64.0, and beta = 110. Crystals of NAD+ synthetase complexed with deamido-NAD+ belong to space group P3(1), with unit cell dimensions of a = b = 63.4, c = 125.7 A
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
60
-
E07950, -
stable at
70
-
E07950, -
40% of the activity at 60C
95
-
-
10 min, inactivation
100
-
-
half-life: 13 min
ORGANIC SOLVENT
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Triton X-100
-
inhibitory effects toward the enzyme are decreased in the presence of Triton X-100
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, recombinant truncated enzyme, overnight, loss of 59% activity
-
-70C, recombinant truncated enzyme, overnight, loss of 30% activity
-
-80 - 4C, recombinant full length enzyme, several months
-
25C, recombinant truncated enzyme, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Ni HiTrap column chromatography, and Superdex-200 gel filtration or Superdex-75 gel filtration
-, Q81RP3
recombinant enzyme from Escherichia coli, over 95% purity
-
recombinantly overexpressed enzyme, to homogeneity
-
native enzyme to homogeneity
E07950, -
recombinant His-tagged enzyme and recombinant thioredoxin-His6-fusion protein from Escherichia coli and insect cells, the His-tag is cleaved off afterwards
-
Ni-NTA Superflow resin chromatography
-
recombinant as His-tagged enzyme from Escherichia coli
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Escherichia coli BL21(DE3)pLysS cells
-, Q81RP3
gene OutB, expression in Escherichia coli BL21(DE3)
-
overexpression in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
-
overexpression in strain BL21(DE3) as HIs-tagged enzyme
-
DNA and amino acid sequence determination and analysis, functional overexpression in Escherichia coli
E07950, -
nadE gene, expression of the enzyme as a His-tagged protein and as a thioredoxin-His6-fusion protein in insect cells of Thrichoplusia ni BTI-TN-5B1-4 via baculovirus infection, for large scale production, and in Escherichia coli
-
expressed in Escherichia coli
-
expressed in Escherichia coli BL21 (DE3) cells
-
functional expression in Escherichia coli strain M-15 as His-tagged enzyme, secretion to the medium
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D593A
-
poor synthetase activity
D593A/F622A
-
synthetase dead
E177A
-
poor synthetase activity
F622A
-
poor synthetase activity
I111A
-
poor synthetase activity
L529A/L604A
-
poor synthetase activity
L604A
-
poor synthetase activity
L604N
-
poor synthetase activity
M621A
-
synthetase mutant that depresses all activities
R112L
-
poor synthetase activity that inhibit substrate synergism
R112S
-
poor synthetase activity that inhibit substrate synergism
Y532A/Y601A
-
synthetase dead
Y601A
-
poor synthetase activity
Y601A/M621A
-
synthetase dead
additional information
-
mutant enzyme with a lower temperature optimum and a non-hyperbolic kinetic versus NH4+
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
pharmacology
-
preparation of isotopically labelled [13N]NAD+, a radiopharmaceutical designed for positron emission tomography, by the NAD+ synthetase immobilized on porous glass beads
analysis
-
the enzyme is used for an enzymatic cycling method for ammonia assay using a system consisting of three enzymes: EC 6.3.1.5, EC 1.1.1.47 and EC 1.6.99.2
analysis
-
method for measurement of allantoin in human serum. Serum allantoin is converted to allantoate by the action of allantoinase,and endogenous ammonia is simultaneously removed by the action of glutamine synthetase II. In the second step, L-methionine sulfoximine is used to inhibit glutamine synthetase II, and ammonia is liberated from allantoate by the activity of allantoate amidohydrolase. The ammonia is then converted to NAD by NAD synthetase. Subsequent action of glucose dehydrogenase and diaphorase acts to cycle the formed NAD between its oxidized and reduced forms, resulting in the production of WST-1 formazan, which is monitored at 450 nm. The assay standard curve is linear from 0 to 70 microM allantoin
analysis
-
use of substrate 2-fluro-ATP as tool for 18F NMR-based activity screening