Information on EC 2.5.1.72 - quinolinate synthase

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

EC NUMBER
COMMENTARY hide
2.5.1.72
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RECOMMENDED NAME
GeneOntology No.
quinolinate synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
glycerone phosphate + iminosuccinate = pyridine-2,3-dicarboxylate + 2 H2O + phosphate
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NAD biosynthesis I (from aspartate)
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nicotine biosynthesis
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superpathway of nicotine biosynthesis
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NAD metabolism
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SYSTEMATIC NAME
IUBMB Comments
glycerone phosphate:iminosuccinate alkyltransferase (cyclizing)
An iron-sulfur protein that requires a [4Fe-4S] cluster for activity [1]. Quinolinate synthase catalyses the second step in the de novo biosynthesis of NAD+ from aspartate in some bacteria, with EC 1.4.3.16 (L-aspartate oxidase) catalysing the first step and EC 2.4.2.19 [nicotinate-nucleotide diphosphorylase (carboxylating)] the third step. In Escherichia coli, two of the residues that are involved in the [4Fe-4S] cluster binding appear to undergo reversible disulfide-bond formation that regulates the activity of the enzyme [5].
CAS REGISTRY NUMBER
COMMENTARY hide
39434-08-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isoform NadA
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
gene nadA
UniProt
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
gene nadA
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
-
three strictly conserved amino acids, Glu198, Tyr109, and Tyr23, are in close proximity to the bound product. Substitution of these amino acids with Gln, Phe, and Phe, respectively, leads to complete loss of activity
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
dihydroxyacetone phosphate + iminoaspartate
? + H2O + phosphate
show the reaction diagram
glycerone phosphate + iminosuccinate
pyridine-2,3-dicarboxylate + 2 H2O + phosphate
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
glycerone phosphate + iminosuccinate
pyridine-2,3-dicarboxylate + 2 H2O + phosphate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S-center
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inhibitor 4,5-dithiohydroxyphthalic acid coordinates to the enzyme [4Fe-4S] cluster through a differentiated iron site
iron-sulfur centre
[4Fe-4S]-center
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NadA contains a [4Fe-4S] cluster cofactor with a unique, non-cysteinyl-ligated, iron ion (Fea), which binds the hydroxyl group of a postulated intermediate in the last step of the reaction to facilitate a dehydration. N1 and the C7 carboxylate group of quinolinate ligate to Fea in a bidentate fashion placing the C5 hydroxyl group of the postulated final intermediate distal to Fea and virtually incapable of coordinating to it
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4Fe-4S cluster
the enzyme requires a [4Fe4S] cluster for full activity
[4Fe-4S] cluster
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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inhibits reactivation of O2-inactivated enzyme
2,2'-dipyridyl
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inhibits reactivation of O2-inactivated enzyme
4,5-dithiohydroxyphthalic acid
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structural analogue of the 5-hydroxy-4,5-dihydropyridine-2,3-dicarboxylic acid intermediate. Compound coordinates to the enzyme [4Fe-4S] cluster through a differentiated iron site, thus leading to the inhibition of quinolate formation. Compound is inhibitory in vitro and in vivo
H2O2
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1 mM, inactivation
paraquat
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inactivation
Phosphoglycolic acid
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.36
dihydroxyacetone phosphate
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25C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.007
4,5-dithiohydroxyphthalic acid
Escherichia coli
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pH not specified in the publication, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.027
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25C, presence of oxygen to reoxidize NadB in the coupled assay
0.05
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25C, presence of fumarate as electron acceptor for NadB in the coupled assay
0.6
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pH 7.0, 25C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36760
x * 36760, calculated
38240
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x * 38246, mass spectrometry, x * 38240, calculated
38246
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x * 38246, mass spectrometry, x * 38240, calculated
41000
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3 * 41000, calculated
80000
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gel filtration, major amount of protein
124000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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plus minor amount of monomer. 2 * 40000, SDS-PAGE, 2 * 39300, calculated
monomer
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1 * 40000, SDS-PAGE, 1 * 39300, calculated
trimer
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3 * 41000, calculated
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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residues C291 and C294 of the C291XXC294XXC297 motif undergo reversible disulfide formation, which regulates the activity of the enzyme. Disulfide-bond formation and reduction are effected by oxidized and reduced forms of thioredoxin, with a midpoint potential of -264 mV for the redox couple
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
both native and SeMet enzyme are crystallized using the hanging-drop vapor-diffusion method at 22C, structure is determined at 2.8 A resolution
in presence of substrate analogue malate. Diffraction to 2.0 A resolution. Triangular architecture composed of a 3fold repeat of three-layer alphabetaalpha sandwich folding. The active site is located at the interface of the three domains and is centered on the pseudo-3fold axis. The malate molecule is tightly held near the bottom of the active site cavity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme, purification from inclusion bodies
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recombinant protein
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recombinant protein, complete loss of activity upon purification of enzyme in aerobic conditions or exposure to oxygen overnight
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Escherichia coli, N-terminal His-tag
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expression with C-terminal His-tag
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expression with N-terminal His-tag
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C110S
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0.4 mol iron per mol of protein, no enzymic activity
C230S
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0.6 mol iron per mol of protein, no enzymic activity
C259S
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4.5 mol iron per mol of protein, 80% of wild-type activity
C318S
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3.3 mol iron per mol of protein, 75% of wild-type activity
C318S/C320S
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0.3 mol iron per mol of protein, no enzymic activity
C320S
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1.5 mol iron per mol of protein, no enzymic activity
C82S
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4.3 mol iron per mol of protein, activity similar to wild-type
C113S
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1.3 iron ions per polypeptide, no catalytic activity
C119A
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2.9 mol of iron and sulfur per mol of protein
C119S
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1.0 iron ions per polypeptide
C128S
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2.7 iron ions per polypeptide
C195S
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1.5 iron ions per polypeptide
C200S
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1.0 iron ions per polypeptide, no catalytic activity
C291A
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3.9 mol of iron and sulfur per mol of protein
C291A/C294A
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3.7 mol of iron and sulfur per mol of protein
C291A/C294A/C297A
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0.5 mol of iron and sulfur per mol of protein
C291S
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0.8 iron ions per polypeptide
C294A
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3.2 mol of iron and sulfur per mol of protein
C294A/C297A
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0.6 mol of iron and sulfur per mol of protein
C294S
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2.1 iron ions per polypeptide
C297S
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0.3 iron ions per polypeptide, no catalytic activity
C64S
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1.4 iron ions per polypeptide
E198Q
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site-directed mutagenesis, inactive mutant
Y109F
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site-directed mutagenesis, inactive mutant
Y23F
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site-directed mutagenesis, inactive mutant
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
O2-dependent inactivation inactivation in extracts can be gradually reversed during anaerobic incubation, but is blocked by 2,2'-dipyridyl or by 1,10-phenanthroline
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
synthesis
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production of quinolinic acid from L-aspartate, dihydroxyacetone phosphate, and O2 by use of enzymes NadA and NadB
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