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 hide
6.3.5.1
-
RECOMMENDED NAME
GeneOntology No.
NAD+ synthase (glutamine-hydrolysing)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + deamido-NAD+ + L-glutamine + H2O = AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acid amide hydrolysis
-
-
carboxylic
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
-
-
NAD biosynthesis from 2-amino-3-carboxymuconate semialdehyde
-
-
NAD biosynthesis I (from aspartate)
-
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NAD metabolism
-
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NAD salvage pathway I
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NAD salvage pathway II
-
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Nicotinate and nicotinamide metabolism
-
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pyridine nucleotide cycling (plants)
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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).
CAS REGISTRY NUMBER
COMMENTARY hide
37318-70-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene nadE
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
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
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
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
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-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
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+ + 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+
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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
ATP + deamido-NAD+ + L-glutamine + H2O
AMP + diphosphate + NAD+ + L-glutamate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
can partially replace Mg2+, at 5 mM 30% of the activity relative to Mg2+
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 hide
LITERATURE
IMAGE
3-[([7-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]heptyl]oxy)carbonyl]-1-methylpyridinium
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-
4-[([7-[5-(benzyloxy)-2-(methoxycarbonyl)-1H-indol-1-yl]heptyl]oxy)carbonyl]-N,N,N-trimethylanilinium
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-
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
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-
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+
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in excess of ATP
Mn2+
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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 hide
LITERATURE
IMAGE
nicotinic acid adenine dinucleotide
-
stimulates activity 50fold
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.015 - 2
ATP
0.017 - 2.9
deamido-NAD+
4.3
deamido-NMN
pH 7.5, 30C
2 - 5
Gln
0.001 - 20
L-Gln
1.1 - 5.3
L-glutamine
0.089 - 23.9
NH3
1.6 - 64.2
NH4+
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.15 - 3
ATP
0.16 - 3.7
deamido-NAD+
0.0032
deamido-NMN
Acinetobacter baumannii
B0V8W9
pH 7.5, 30C
0.012 - 3
L-Gln
0.12 - 6.4
L-glutamine
0.0045 - 3.3
NH3
additional information
additional information
Saccharomyces cerevisiae
-
turnover number: 1260, NAD+ production
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6 - 5
ATP
3.7 - 34
deamido-NAD+
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 - 0.42
L-Glu
0.051 - 0.7
NH3
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 hide
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 hide
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 hide
LITERATURE
0.0002
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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
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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
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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
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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
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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
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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
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recombinant NadE-738, nitrogen donor glutamine
0.0533
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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
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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
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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
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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
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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
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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
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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 hide
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
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with NH4+ as amide donor
8.5
-
recombinant NadE-738, nitrogen donor NH4+, recombinant NadE-679, nitrogen donor glutamine
8.8
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recombinant NadE-738, nitrogen donor glutamine
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8
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6.0: about 75% of maximal activity, 8.0: about 55% of maximal activity
7.8 - 9.3
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7.8: about 40% of maximal activity, 9.3: about 50% of maximal activity, with NH4+ as amide donor
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
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 hide
LITERATURE
65 - 80
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at temperatures above 65C the glutamine-dependent reaction becomes about 20% faster than the ammonia-dependent at 80C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
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
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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
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traces
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 hide
LITERATURE
483000
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gel filtration
500000
non-denaturing PAGE
600000
-
gel filtration, NadE-679
630000
-
high speed equilibrium ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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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 hide
LITERATURE
6 - 10
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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, 0.2 mg per ml, 10 d, 60% loss of activity
-
4C, 24 h, complete loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant enzymes expressed in Escherichia coli
-
partial
recombinant enzyme
-
recombinant NAD+ synthase, Ni2+ chelate resin affinity chromatography
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recombinant NadE-738 and NadE-679, Ni2+-nitrilotriacetic acid agarose, gel filtration
-
using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
-
expressed in Escherichia coli as a His-tagged fusion protein
expression in Escherichia coli
-
expression in Escherichia coli, with SUMO tag
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expression in Sf9 insect cells
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expression of His-tagged NAD+ synthase in Escherichia coli
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expression of Nadsyn1 and mutant enzyme C175S as His6-tagged proteins in COS-7 cells
most efficient expression of His-tagged NadE-738 and NadE-679 in Escherichia coli strain Origami
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
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site-directed mutagenesis, inactive mutant, analysis of ligand binding structures
-
L486A
-
site-directed mutagenesis
-
L486F
-
site-directed mutagenesis
-
Y58A
-
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 hide
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
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