Information on EC 1.17.1.5 - nicotinate dehydrogenase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.17.1.5
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RECOMMENDED NAME
GeneOntology No.
nicotinate dehydrogenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
nicotinate + H2O + NADP+ = 6-hydroxynicotinate + NADPH + H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of amide bond
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hydroxylation
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oxidation
redox reaction
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Microbial metabolism in diverse environments
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Nicotinate and nicotinamide metabolism
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nicotinate degradation III
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SYSTEMATIC NAME
IUBMB Comments
nicotinate:NADP+ 6-oxidoreductase (hydroxylating)
A flavoprotein containing non-heme iron. The enzyme is capable of acting on a variety of nicotinate analogues to varying degrees, including pyrazine-2-carboxylate, pyrazine 2,3-dicarboxylate, trigonelline and 6-methylnicotinate. The enzyme from Clostridium barkeri also possesses a catalytically essential, labile selenium that can be removed by reaction with cyanide.
CAS REGISTRY NUMBER
COMMENTARY hide
9059-03-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
DSM 6335
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Manually annotated by BRENDA team
Bacillus sp. DSM 2923, strain AS1 isolated from the culture obtained by DSM
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
nicotinic acid-fermenting
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Manually annotated by BRENDA team
strain TN5
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Manually annotated by BRENDA team
strain TN5
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Manually annotated by BRENDA team
strain KT2440
SwissProt
Manually annotated by BRENDA team
strain IFO 12648
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Manually annotated by BRENDA team
strain IFO 12648
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
2,3-pyrazinedicarboxylate + H2O + NADP+
?
show the reaction diagram
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10-20% of the activity with nicotinate
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?
2-hydroxynicotinate + H2O + electron acceptor
?
show the reaction diagram
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hydroxylation at a low rate
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?
2-pyrazinecarboxylate + H2O + electron acceptor
?
show the reaction diagram
2-pyrazinecarboxylate + H2O + NADP+
?
show the reaction diagram
3,5-pyridinedicarboxylate + H2O + NADP+
?
show the reaction diagram
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5-10% of the activity with nicotinate
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?
3-cyanopyridine + H2O + electron acceptor
?
show the reaction diagram
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hydroxylation at a low rate
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?
3-cyanopyridine + H2O + NADP+
6-hydroxy-3-cyanopyridine + NADPH + H+
show the reaction diagram
3-pyridinesulfonate + H2O + electron acceptor
?
show the reaction diagram
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?
6-methylnicotinate + H2O + NADP+
?
show the reaction diagram
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5-10% of the activity with nicotinate
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?
nicotinate + H2O + electron acceptor
6-hydroxynicotinate + reduced electron acceptor
show the reaction diagram
nicotinate + H2O + NADP+
6-hydroxynicotinate + NADPH
show the reaction diagram
nicotinate + H2O + NADP+
6-hydroxynicotinate + NADPH + H+
show the reaction diagram
nicotinate + H2O + phenazine methosulfate
6-hydroxynicotinate + reduced phenazine methosulfate
show the reaction diagram
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?
picolinic acid + NADP+ + H2O
6-hydroxypicolinic acid + NADP+
show the reaction diagram
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pyridine-3-carboxylate + NADP+ + H2O
6-hydroxy-pyridine-3-carboxylate + NADPH + H+
show the reaction diagram
trigonelline + H2O + NADP+
?
show the reaction diagram
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5-10% of the activity with nicotinate
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?
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
3-cyanopyridine + H2O + NADP+
6-hydroxy-3-cyanopyridine + NADPH + H+
show the reaction diagram
nicotinate + H2O + electron acceptor
6-hydroxynicotinate + reduced electron acceptor
show the reaction diagram
nicotinate + H2O + NADP+
6-hydroxynicotinate + NADPH
show the reaction diagram
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first step in the anaerobic decomposition of nicotinate, NADP+ may be the natural, primary electron acceptor
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?
nicotinate + H2O + NADP+
6-hydroxynicotinate + NADPH + H+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4Fe-4S-center
Bactopterin
benzyl viologen
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can replace NADP+
flavin
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primary electron acceptor, 1.5-2 mol tightly bound flavin per mol of enzyme
molybdenum cofactor
molybdopterin
NADP+
NADPH
phenazine methosulfate
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phenazine methosulfate-requiring enzyme
additional information
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
the enzyme contains a [2Fe2S] domain, a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, overview; the enzyme contains a [2Fe2S] domain, a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, overview; the enzyme contains a [2Fe2S] domain, a type II [2Fe-2S] motif and a type I [2Fe-2S] motif, overview
additional information
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enzyme activity increases with increasing concentrations of molybdate in growth medium
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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1 mM, 30-50% inhibition
2,2'-dipyridyl
6-Hydroxynicotinate
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inhibits effectively
Acriflavin
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1 mM, 10 min at 35C, 22% loss of activity
AgNO3
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1 mM, 10 min at 35C, complete loss of activity
Atabrine
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1 mM, 30-50% inhibition
CuCl2
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1 mM, 10 min at 35C, complete loss of activity
HgCl2
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1 mM, 10 min at 35C, complete loss of activity
N-ethylmaleimide
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1 mM, 10 min at 35C, complete loss of activity
NaCl
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sufficient NaCl inhibits enzyme activity significantly
NaN3
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1 mM, 10 min at 35C, 17% loss of activity
p-chloromercuribenzoate
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1 mM, 10 min at 35C, complete loss of activity
phenylhydrazine
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1 mM, 10 min at 35C, 15% loss of activity
Selenophosphate
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7 mM, 30 min, anaerobic conditions, reversible complete inactivation, time-dependent
sodium selenide
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7 mM, 10 min, anaerobic conditions, reversible complete inactivation, time-dependent
Sulfide
additional information
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not inhibited by incubation for 1 h at room temperature with 100 mM KCN
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
arsenate
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activates less than phosphate
methylphosphate
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activates less than phosphate
phosphate
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slight activation, half-maximal activation at 50 mM
additional information
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not activated by high concentrations of citrate, sulfate, or diphosphate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.1
2-Hydroxynicotinate
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9.8
2-pyrazinecarboxylate
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150
3-Cyanopyridine
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0.7
3-pyridinesulfonate
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0.028
NADP+
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0.07 - 1
nicotinate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.83
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1
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2-hydroxynicotinate
2.2
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crude extract, high activity achieved if nicotinate is the only substrate
4.4
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purified enzyme, Difco yeast extract without selenium as growth medium
12
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3-cyanopyridine
20
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purified enzyme, average value, Difco yeast extract with selenium as growth medium
104
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3-pyridinesulfonate
170
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2-pyrazinecarboxylate
672
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nicotinate
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8
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phosphate buffer
8 - 8.5
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triethanolamine buffer
8
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triethanolamine phosphate buffer
8.3 - 9
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Tris buffer or diphosphate buffer
8.5
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dimethylglutarate-phosphate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9
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broad pH-range with maximum at pH 8.3
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
additional information
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assay at room temperature
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
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x * 85000 + x * 34000 + x * 20000, additional bands with increasing period of storage, subunits are loosely linked by electrostatic interactions, SDS-PAGE
20981
x * 20981, NdhS, sequence calculation
33000
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1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
34000
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x * 85000 + x * 34000 + x * 20000, additional bands with increasing period of storage, subunits are loosely linked by electrostatic interactions, SDS-PAGE
46042
x * 46042, NdhM, sequence calculation
80000
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1 * 80000, SDS-PAGE, in presence of 50 mM 2-mercaptoethanol
82389
x * 82389, NdhL, sequence calculation
85000
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x * 85000 + x * 34000 + x * 20000, additional bands with increasing period of storage, subunits are loosely linked by electrostatic interactions, SDS-PAGE
100000
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gel filtration
130000
SDS-PAGE
160000
280000 - 340000
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gel filtration, non-denaturing PAGE
300000
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analytical ultracentrifugation
400000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
monomer
octamer
dimer of heterotetramers, subunits of the heterotetramer: 1 * 50000 + 1 * 37000 + 1* 33000 + 1 * 23000, X-ray diffraction
oligomer
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x * 85000 + x * 34000 + x * 20000, additional bands with increasing period of storage, subunits are loosely linked by electrostatic interactions, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8.8
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10 min at 50C in various buffers, stable
644529
8
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most stable at alkaline pH
644535
additional information
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maximal stability during incubation for 14 h at room temperature, after 9 days storage at pH 8, 25% of the initial activity retained
644535
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
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10 min in 0.1 M potassium phosphate buffer, pH 7, stable below
additional information
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hydroxylase activity is more sensitive to heat inactivation than are diaphorase and NADPH-oxidase activities
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme is most stable at alkaline pH in the presence of glycerol, 20% glycerol and 400 mM KCl stabilize
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lyophilization completely inactivates, high protein concentration may have a stabilizing effect on enzyme activity, sugar protects enzyme from dissociation
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membrane-bound enzyme is stable, but solubilized enzyme is very labile, 20 mM nicotinate stabilizes
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
exposure of substrate-reduced enzyme to air results in a complete loss of activity, enzyme before reduction is much less sensitive to oxygen inactivation
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644535
oxygen-sensitive enzyme
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644528
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, increase of activity in crude extract
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room temperature, 50 mM Tris-HCl buffer, pH 8.2, after 1 day 40% loss of hydroxylase activity, after 7 days 62% loss of hydroxylase activity, NADPH oxidase and diaphorase activity of enzyme are more stable
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room temperature, pH8, 9 days, 75% loss of hydroxylase activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
112fold purification
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126fold purification
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24.4fold purification
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24fold increase in specific activity
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partial purification
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Pseudomonas entomophila, subcloning of mutants in Escherichia coli failed
gene ndhL, belongs to the ndh cluster with genes ndhS and ndhM, DNA and amino acid sequence determination, sequence comparison, recombinant expression of the nicotinate dehydrogenase gene cluster as His-tagged proteins in Pseudomonas entomophila strain L48, subcloning in Escherichia coli strain DH10B; gene ndhM, belongs to the ndh cluster with genes ndhL and ndhS, DNA and amino acid sequence determination, sequence comparison, recombinant expression of the nicotinate dehydrogenase gene cluster as His-tagged proteins in Pseudomonas entomophila strain L48, subcloning in Escherichia coli strain DH10B; gene ndhS, belongs to the ndh cluster with genes ndhL and ndhM, DNA and amino acid sequence determination, sequence comparison, recombinant expression of the nicotinate dehydrogenase gene cluster as His-tagged proteins in Pseudomonas entomophila strain L48, subcloning in Escherichia coli strain DH10B
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
after denaturation by acid precipitation at pH 4.5 with HCl, recombining and neutralizing of precipitated protein and yellow supernatant results in a reconstitution of over 90% of the original activity
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