Information on EC 6.3.5.1 - NAD+ synthase (glutamine-hydrolysing)

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

EC NUMBER
COMMENTARY
6.3.5.1
-
RECOMMENDED NAME
GeneOntology No.
NAD+ synthase (glutamine-hydrolysing)
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + deamido-NAD+ + L-glutamine + H2O = AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Acid amide hydrolysis
-
-
carboxylic
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NAD biosynthesis from 2-amino-3-carboxymuconate semialdehyde
-
-
NAD biosynthesis I (from aspartate)
-
-
NAD salvage pathway I
-
-
pyridine nucleotide cycling (plants)
-
-
NAD metabolism
-
-
Nicotinate and nicotinamide metabolism
-
-
Metabolic pathways
-
-
SYSTEMATIC NAME
IUBMB Comments
Deamido-NAD+:L-glutamine amido-ligase (AMP-forming)
NH3 can act instead of glutamine (cf. EC 6.3.1.5 NAD+ synthase).
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
abNadE
-
enzyme possesses an additional glutamine transferase domain
General stress protein 38
-
-
-
-
glutamine-dependent NAD+ synthetase
-
glutamine-dependent NAD+ synthetase
Mycobacterium tuberculosis ATCC 256118
-
-
glutamine-dependent NAD+ synthetase
-
-
GSP38
-
-
-
-
mtuNadE
Mycobacterium tuberculosis ATCC 256118
-
-
NAD synthetase (glutamine)
-
-
-
-
NAD(+) synthase [glutamine-hydrolyzing]
-
-
-
-
NAD+ synthetase
-
-
NAD+ synthetase
Mycobacterium tuberculosis ATCC 256118
-
-
NAD+ synthetase (glutamine-hydrolysing)
-
-
-
-
NadE-679
-
short NadE form, probably raising from translation of NadE gene from an internal start codon
Nicotinamide adenine dinucleotide synthetase (glutamine)
-
-
-
-
Nitrogen-regulatory protein
-
-
-
-
Qns1
-
couples a glutamine amidotransferase domain to a second active site that requires ammonia as a reactant
Sporulation protein outB
-
-
-
-
Synthetase, nicotinamide adenine dinucleotide (glutamine)
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37318-70-0
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
recombinant enzyme
-
-
Manually annotated by BRENDA team
NAD+ synthase is encoded by the MTCY428.08 gene
-
-
Manually annotated by BRENDA team
Mycobacterium tuberculosis ATCC 256118
gene nadE
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
most residues lining the ATP-binding site are conserved among the glutamine-dependent NAD+ synthetases, whereas residues not conserved, such as Leu399 andGly366, interact with the adenine ring and the adenylyl ribose with the backbone oxygen and nitrogen
evolution
Mycobacterium tuberculosis ATCC 256118
-
most residues lining the ATP-binding site are conserved among the glutamine-dependent NAD+ synthetases, whereas residues not conserved, such as Leu399 andGly366, interact with the adenine ring and the adenylyl ribose with the backbone oxygen and nitrogen
-
malfunction
nadE is dispensable when the nondeamidating salvage pathway functions as the only route of NAD biogenesis
malfunction
-
a homolog of the human SIRT6-like gene, SRT2, is upregulated in the NAD+ synthase mutant, which shows a longer vegetative life span than wild-type cells
physiological function
nadE, encoding glutamine-dependent NAD synthetase, is dispensable when the nondeamidating salvage pathway of nicotinamide salvage/recycling functions as the only route of NAD biogenesis
metabolism
due to the presence of an additional glutamine transferase domain, abNadE can efficiently utilize l-glutamine (as well as ammonia) for the amidation of NAD precursor
additional information
the enzyme contains glutaminase and synthetase active sites, structures and ligand binding, overview. The ATP-binding site is located in a deep cleft formed solely by a single subunit next to the nicotinic acidadenine dinucleotide-binding site
additional information
Mycobacterium tuberculosis ATCC 256118
-
the enzyme contains glutaminase and synthetase active sites, structures and ligand binding, overview. The ATP-binding site is located in a deep cleft formed solely by a single subunit next to the nicotinic acidadenine dinucleotide-binding site
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + deamido-NAD+ + Asn
AMP + diphosphate + NAD+ + Asp
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + Gln + hydroxylamine
AMP + diphosphate + hydroxamate analog + ?
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-Gln
AMP + diphosphate + NAD+ + L-Glu
show the reaction diagram
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
last step in NAD+ biosynthesis
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
NADsyn1 uses not only ammonia but also glutamine. The Vmax/KM-value for NADsyn1 with glutamine is 6.3fold higher than the Vmax/Km-value for NH3
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
Mycobacterium tuberculosis ATCC 256118
-
-
?
ATP + deamido-NAD+ + NH3
AMP + diphosphate + NAD+
show the reaction diagram
NADsyn1 uses not only ammonia but also glutamine. The Vmax/KM-value for NADsyn1 with glutamine is 6.3fold higher than the Vmax/Km-value for NH3
-
?
ATP + deamido-NAD+ + NH3 + H2O
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3 + H2O
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH3 + H2O
AMP + diphosphate + NAD+
show the reaction diagram
-
-
?
ATP + deamido-NAD+ + NH3 + H2O
AMP + diphosphate + NAD+
show the reaction diagram
-
NAD+ synthase is able to use ammonia or glutamine as nitrogen source
-
?
ATP + deamido-NAD+ + NH4+
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
-
ATP + deamido-NAD+ + NH4+
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + NH4+ + H2O
AMP + diphosphate + NAD+
show the reaction diagram
-
-
-
?
ATP + deamido-NMN + L-Gln
AMP + diphosphate + NMN + L-Glu
show the reaction diagram
poor substrate
-
?
Gln + hydroxylamine
AMP + diphosphate + hydroxamate analog + ?
show the reaction diagram
-
-
-
-
additional information
?
-
-
no activity with NH4+ as nitrogen donor at 3 mM NH4+
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
P9WJJ3
-
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
last step in NAD+ biosynthesis
-
?
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
Mycobacterium tuberculosis ATCC 256118
P9WJJ3
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ATP
structure of the ATP-binding site at the mtuNadE synthetase domain, overview
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
-
can partially replace Mg2+, at 5 mM 30% of the activity relative to Mg2+
K+
-
Km: 8.32 mM; required
Mg2+
-
required
Mg2+
-
required
Mg2+
-
Km: 1.36 mM; required
Mg2+
required for activity
Mn2+
-
can partially replace Mg2+ at 1 mM. At 5 mM inhibition of NAD+ formation
NH4+
-
as effective as K+ in stimulating activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3-[([7-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]heptyl]oxy)carbonyl]-1-methylpyridinium
-
-
4-[([7-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]heptyl]oxy)carbonyl]-N,N,N-trimethylanilinium
-
-
4-[([8-[5-(benzyloxy)-1H-indol-1-yl]octyl]oxy)carbonyl]-N,N,N-trimethylanilinium
-
-
4-[2-([9-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]nonyl]oxy)-2-oxoethyl]-N,N,N-trimethylanilinium
-
-
6-diazo-5-oxo-L-norleucine
-
-
adenosine 5'-[alpha,beta-methylene]triphosphate
i.e. AMP-CPP, nonhydrolysable analogue of ATP
azaserine
-
progressively inhibits glutamine-dependent activity. With NH4+ as amide donor there is an initial 20% inhibition, then the NH4+-dependent activity remains constant
diisopropyl fluorophosphate
-
-
gossypol
-
-
Mg2+
-
in excess of ATP
Mn2+
-
can partially replace Mg2+ at 1 mM. At 5 mM inhibition of NAD+ formation
nicotinic acid-adenine dinucleotide
i.e. NaAD+
p-chloromercuribenzoate
-
glutamine-dependent and NH4+-dependent activity
Pb2+
-
-
Zn2+
-
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
nicotinic acid adenine dinucleotide
-
stimulates activity 50fold
additional information
the formation of the synthetase intermediate analogues complex, NaAD-AMP intermediate and diphosphatei, triggers structural changes leading to enzyme activation
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.015
ATP
-
pH 7.5
0.089
ATP
37C, wild-type NADsyn1
0.095
ATP
-
pH 8.3, 37C
0.1 - 2
ATP
pH and temperature not specified in the publication
0.12
ATP
37C, NADsyn1 C175S mutant
0.12
ATP
-
wild-type, glutamine-dependent reaction
0.154
ATP
-
-
0.17
ATP
-
pH 7.6
0.18
ATP
-
-
0.6
ATP
-
mutant C176A, ammonia-dependent reaction
0.79
ATP
-
37C, pH 8.0, recombinant NadE-679
0.8
ATP
-
mutant enzyme E52A; wild-type enzyme
1.3
ATP
-
37C, pH 8.5, recombinant NadE-738
1.3
ATP
-
mutant enzyme C176A
1.4
ATP
-
wild-type, ammonia-dependent reaction
0.017
deamido-NAD+
pH 7.5, 30C; pH 7.5, 30C
0.021
deamido-NAD+
-
pH 8.3, 37C
0.04
deamido-NAD+
-
pH 7.5
0.049
deamido-NAD+
37C, wild-type NADsyn1
0.083
deamido-NAD+
-
-
0.108
deamido-NAD+
-
-
0.13
deamido-NAD+
-
wild-type, glutamine-dependent reaction
0.19
deamido-NAD+
-
pH 7.6
0.2
deamido-NAD+
37C, NADsyn1 C175S mutant
0.5
deamido-NAD+
-
wild-type enzyme
0.52
deamido-NAD+
-
37C, pH 8.0, recombinant NadE-679
0.65
deamido-NAD+
-
mutant C176A, ammonia-dependent reaction
0.7
deamido-NAD+
-
wild-type, ammonia-dependent reaction
1.4
deamido-NAD+
-
mutant enzyme C176A; mutant enzyme E52A
2.9
deamido-NAD+
-
37C, pH 8.5, recombinant NadE-738
4.3
deamido-NMN
pH 7.5, 30C
2.17
Gln
-
-
0.001
L-Gln
-
pH 7.5
0.42
L-Gln
-
pH 8.3, 37C
1.3
L-Gln
-
wild-type, glutamine-dependent reaction
1.6
L-Gln
-
wild-type enzyme
7.8
L-Gln
-
mutant D656A, glutamine-dependent reaction
20
L-Gln
-
wild-type, ammonia-dependent reaction
1.1
L-glutamine
-
37C, pH 8.8, recombinant NadE-738
1.4
L-glutamine
-
mutant enzyme Y601A, at 37C
1.44
L-glutamine
37C, wild-type NADsyn1
1.5
L-glutamine
pH and temperature not specified in the publication
1.6
L-glutamine
-
37C, pH 8.5, recombinant NadE-679
3.5
L-glutamine
-
mutant enzyme Y532A/Y601A, at 37C
5.3
L-glutamine
-
wild type enzyme, at 37C
0.089
NH3
-
mutant D656A, ammonia-dependent reaction
0.33
NH3
-
pH 8.3, 37C
13.1
NH3
37C, wild-type NADsyn1
15
NH3
-
mutant C176A, ammonia-dependent reaction
23.9
NH3
37C, NADsyn1 C175S mutant
1.6
NH4+
-
mutant enzyme E52A
2.3
NH4+
-
mutant enzyme C176A
2.5
NH4+
-
37C, pH 8.0, recombinant NadE-679
2.7
NH4+
-
wild-type enzyme
6.4
NH4+
-
pH 8.6
27
NH4+
-
37C, pH 8.5, recombinant NadE-738
64.2
NH4+
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.15
ATP
Thermotoga maritima
-
pH 8.3, 37C
0.6
ATP
Mycobacterium tuberculosis
-
wild-type, glutamine-dependent reaction
2.9
ATP
Mycobacterium tuberculosis
-
wild-type, ammonia-dependent reaction
3
ATP
Mycobacterium tuberculosis
-
mutant C176A, ammonia-dependent reaction
0.16
deamido-NAD+
Thermotoga maritima
-
pH 8.3, 37C
0.5
deamido-NAD+
Mycobacterium tuberculosis
-
wild-type, glutamine-dependent reaction
0.57
deamido-NAD+
Acinetobacter baumannii
B0V8W9
pH 7.5, 30C; pH 7.5, 30C
2.6
deamido-NAD+
Mycobacterium tuberculosis
-
wild-type, ammonia-dependent reaction
3.7
deamido-NAD+
Mycobacterium tuberculosis
-
mutant C176A, ammonia-dependent reaction
0.0032
deamido-NMN
Acinetobacter baumannii
B0V8W9
pH 7.5, 30C
0.012
L-Gln
Mycobacterium tuberculosis
-
mutant D656A, glutamine-dependent reaction
0.288
L-Gln
Thermotoga maritima
-
pH 8.3, 37C
0.55
L-Gln
Mycobacterium tuberculosis
-
wild-type, glutamine-dependent reaction
3
L-Gln
Mycobacterium tuberculosis
-
wild-type, ammonia-dependent reaction
0.12
L-glutamine
Saccharomyces cerevisiae
-
mutant enzyme Y532A/Y601A, at 37C
0.16
L-glutamine
Saccharomyces cerevisiae
-
mutant enzyme Y601A, at 37C
0.0045
NH3
Mycobacterium tuberculosis
-
mutant D656A, ammonia-dependent reaction
0.24
NH3
Thermotoga maritima
-
pH 8.3, 37C
3.3
NH3
Mycobacterium tuberculosis
-
mutant C176A, ammonia-dependent reaction
6.4
L-glutamine
Saccharomyces cerevisiae
-
wild type enzyme, at 37C
additional information
additional information
Saccharomyces cerevisiae
-
turnover number: 1260, NAD+ production
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.6
ATP
Thermotoga maritima
-
pH 8.3, 37C
4
2.1
ATP
Mycobacterium tuberculosis
-
wild-type, ammonia-dependent reaction
4
5
ATP
Mycobacterium tuberculosis
-
mutant C176A, ammonia-dependent reaction; wild-type, glutamine-dependent reaction
4
3.7
deamido-NAD+
Mycobacterium tuberculosis
-
wild-type, ammonia-dependent reaction
1300
3.8
deamido-NAD+
Mycobacterium tuberculosis
-
wild-type, glutamine-dependent reaction
1300
5.7
deamido-NAD+
Mycobacterium tuberculosis
-
mutant C176A, ammonia-dependent reaction
1300
7.6
deamido-NAD+
Thermotoga maritima
-
pH 8.3, 37C
1300
34
deamido-NAD+
Acinetobacter baumannii
B0V8W9
pH 7.5, 30C; pH 7.5, 30C
1300
0.00074
deamido-NMN
Acinetobacter baumannii
B0V8W9
pH 7.5, 30C
14943
0.68
L-Gln
Thermotoga maritima
-
pH 8.3, 37C
337
0.0015
L-Glu
Mycobacterium tuberculosis
-
mutant D656A, glutamine-dependent reaction
202
0.15
L-Glu
Mycobacterium tuberculosis
-
wild-type, ammonia-dependent reaction
202
0.051
NH3
Mycobacterium tuberculosis
-
mutant D656A, ammonia-dependent reaction
27
0.22
NH3
Mycobacterium tuberculosis
-
mutant C176A, ammonia-dependent reaction
27
0.7
NH3
Thermotoga maritima
-
pH 8.3, 37C
27
0.42
L-Glu
Mycobacterium tuberculosis
-
wild-type, glutamine-dependent reaction
202
additional information
additional information
Homo sapiens
Q6IA69
the Vmax/KM-value for NADsyn1 with glutamine is 6.3fold higher than the Vmax/Km-value for NH3
2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.7
adenosine 5'-[alpha,beta-methylene]triphosphate
pH and temperature not specified in the publication
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0633
3-[([7-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]heptyl]oxy)carbonyl]-1-methylpyridinium
Mycobacterium tuberculosis
-
pH 8.5, 37C
0.0218
4-[([7-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]heptyl]oxy)carbonyl]-N,N,N-trimethylanilinium
Mycobacterium tuberculosis
-
pH 8.5, 37C
0.0248
4-[([8-[5-(benzyloxy)-1H-indol-1-yl]octyl]oxy)carbonyl]-N,N,N-trimethylanilinium
Mycobacterium tuberculosis
-
pH 8.5, 37C
0.0578
4-[2-([9-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]nonyl]oxy)-2-oxoethyl]-N,N,N-trimethylanilinium
Mycobacterium tuberculosis
-
pH 8.5, 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.0002
-
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
0.0017
-
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; mutant enzyme Y532A/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
0.0087
-
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
0.015
-
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
0.017
-
-
0.024
-
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
-
recombinant NadE-738, nitrogen donor glutamine
0.0533
-
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
0.09
-
glutamine-dependent activity
0.0931
-
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
-
recombinant NadE-738, nitrogen donor NH4+
0.112
-
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
0.23
-
NH3 dependent activity
0.288
-
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
0.293
-
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
0.435
-
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
0.44
-
recombinant NadE-679, nitrogen donor glutamine
0.644
-
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
0.67
-
recombinant NadE-679, nitrogen donor NH4+
2.33
-
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
additional information
-
coupled enzyme assay for the measurement of recombinant human NAD+ synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from reazurin via diaphorase
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.2 - 8.4
-
broad peak of activity with a maximum around 7.6, with Gln as the amide donor
6.8 - 7.4
-
-
8
-
recombinant NadE-679, nitrogen donor NH4+
8.4 - 8.8
-
with NH4+ as amide donor
8.5
-
recombinant NadE-738, nitrogen donor NH4+, recombinant NadE-679, nitrogen donor glutamine
8.8
-
recombinant NadE-738, nitrogen donor glutamine
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 8
-
6.0: about 75% of maximal activity, 8.0: about 55% of maximal activity
7.8 - 9.3
-
7.8: about 40% of maximal activity, 9.3: about 50% of maximal activity, with NH4+ as amide donor
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
80
-
at temperatures above 65C the glutamine-dependent reaction becomes about 20% faster than the ammonia-dependent at 80C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
65 - 80
-
at temperatures above 65C the glutamine-dependent reaction becomes about 20% faster than the ammonia-dependent at 80C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
-
wild-type Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I show different NADS mRNA expression pattern
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
the major sites of NADsyn1 gene expression are the small intestine, kidney, liver, and testis, whereas the skeletal muscle, spleen, lung, heart, and brain show a weak signal
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Cytophaga hutchinsonii (strain ATCC 33406 / NCIMB 9469)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
483000
-
gel filtration
1626
500000
non-denaturing PAGE
652364
600000
-
gel filtration, NadE-679
653750
630000
-
high speed equilibrium ultracentrifugation
1625
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
-
x * 65000 + x * 80000, SDS-PAGE
?
-
x * 98000, SDS-PAGE
hexamer
6 * 80000, wild-type and C175S mutant enzyme, SDS-PAGE; 6 * 80300, calculated from sequence
octamer
-
8 * 67000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme and selenomethionine-derivative, to 2.35 and 3.0 A resolution, respectively. Homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40 A intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites
-
wild-type mtuNadE in complex with 6-diazo-5-oxo-L-norleucine/NaAD+ and mutant enzymes, hanging drop vapour diffusion method, different combinations of the ligands 3 mM NaAD+, 4 mM ATP, 7 mM adenosine 5'-[alpha,beta-methylene]triphosphate, 20 mM L-glutamine, 6 mM NAD+, 10 mM AMP, 4 mM diphosphate, 100 mM L-glutamate, and 10 mM or 120 mM MgCl2 are used in the co-crystallization experiments, crystallization solution contains 1.2-1.6 M ammonium citrate tribasic, pH 6.5-8.0, with 5-15% v/v glycerol, 20C, X-ray diffraction structure determination analysis at 2.0-2.85 A resolution, molecular replacement
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 10
-
overnight, no loss of activity
1625
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
EDTA and KCl increase stability substantially
-
repeated freezing and thawing accelerates inactivation
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, 24 h, complete loss of activity
-
4C, 0.2 mg per ml, 10 d, 60% loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
using Ni-NTA chromatography
partial
-
mutant enzymes expressed in Escherichia coli
-
recombinant NAD+ synthase, Ni2+ chelate resin affinity chromatography
-
recombinant NadE-738 and NadE-679, Ni2+-nitrilotriacetic acid agarose, gel filtration
-
recombinant enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli as a His-tagged fusion protein
expression in Sf9 insect cells
-
expression of Nadsyn1 and mutant enzyme C175S as His6-tagged proteins in COS-7 cells
expression in Escherichia coli
-
expression of His-tagged NAD+ synthase in Escherichia coli
-
most efficient expression of His-tagged NadE-738 and NadE-679 in Escherichia coli strain Origami
-
expressed in Escherichia coli
-
expression in Escherichia coli, with SUMO tag
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
S740I
-
Chlamydomonas cells bearing a NAD+ synthase mutation have a defective NAD+ de novo synthesis pathway. Mutant cells show longer vegitative life span than wild-type cells. Up-regulation of a sirtuin gene is linked to the extended life span in the mutant strain
S740I
-
mutant fails to grow on Sager and Granick rich medium without nicotinamide or Sager and Garnick medium supplemented with 3-acetylpyridine. Mutants grows well on media supplied with either nicotinamide or NMN. Addition of nicotinic acid shows very weak rescue of the nic- mutant phenotype. Addition of 3-hydroxyanthranilate cannot rescue the growth defect. Sirtuin SRT2 levels are 2- to 2.5fold increased in the mutant with concomitant increase in reproductive capactiy
C175S
in contrast with the wild-type NADsyn1, the activity of the mutant NADsyn1 (C175S-NADsyn1) is not detected when glutamine is used as a substrate, whereas the activity remains unaltered with NH4Cl
C176A
-
complete loss of glutamine-dependent activity, retains 90% of its NH3-dependent activity
C176A
-
activity smilar to wild-type
C176A
site-directed mutagenesis, inactive mutant, analysis of ligand binding structures
D656A
-
strong decrease in catalytic efficiency
E52A
-
complete loss of glutamine-dependent activity, retains 30% of its NH3-dependent activity
K121A
-
complete loss of glutamine-dependent and NH3-dependent activity
L486A
site-directed mutagenesis
L486F
site-directed mutagenesis
Y58A
site-directed mutagenesis
C176A
Mycobacterium tuberculosis ATCC 256118
-
site-directed mutagenesis, inactive mutant, analysis of ligand binding structures
-
L486A
Mycobacterium tuberculosis ATCC 256118
-
site-directed mutagenesis
-
L486F
Mycobacterium tuberculosis ATCC 256118
-
site-directed mutagenesis
-
Y58A
Mycobacterium tuberculosis ATCC 256118
-
site-directed mutagenesis
-
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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
-
erythrocyte NAD synthetase is activated by lead, and the activity is a sensitive indicator of lead exposure in human
synthesis
-
enzymatic method for efficient synthesis of NAD+ of high purity with 3H, 14C, or other labels at any nonexchangeable position of the NMN+ or AMP portions of the NAD+ molecule