Information on EC 1.13.11.9 - 2,5-dihydroxypyridine 5,6-dioxygenase

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The expected taxonomic range for this enzyme is: Proteobacteria

EC NUMBER
COMMENTARY
1.13.11.9
-
RECOMMENDED NAME
GeneOntology No.
2,5-dihydroxypyridine 5,6-dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
2,5-dihydroxypyridine + O2 = N-formylmaleamic acid
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Microbial metabolism in diverse environments
-
Nicotinate and nicotinamide metabolism
-
nicotinate degradation I
-
nicotine degradation II
-
SYSTEMATIC NAME
IUBMB Comments
2,5-dihydroxypyridine:oxygen 5,6-oxidoreductase
Requires Fe2+.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2,5-DHP dioxygenase
-
-
2,5-dihydroxypyridine dioxygenase
-
-
2,5-dihydroxypyridine oxygenase
-
-
-
-
2,5DHP dioxygenase
-
-
EC 1.13.1.9
-
-
formerly
-
NicX protein
-
-
oxygenase, 2,5-dihydroxypyridine 5,6-di-
-
-
-
-
pyridine-2,5-diol dioxygenase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9029-57-6
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
G2 and 2L
-
-
Manually annotated by BRENDA team
Gram-negative rod
-
-
-
Manually annotated by BRENDA team
KT2440; strain KT2440
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,5-dihydroxypyridine + O2
N-formylmaleamic acid
show the reaction diagram
-
-
-
-
?
2,5-dihydroxypyridine + O2
N-formylmaleamic acid
show the reaction diagram
-
aerobic catabolism of nicotinic acid, extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
-
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
-
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
-
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
strictly specific for 2,5-dihydroxypyridine
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
Gram-negative rod
-
O2 cannot be replaced by methylene blue
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
nicotinic acid catabolism
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
nicotinic acid catabolism
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
Pseudomonas fluorescens N-9
-
-, nicotinic acid catabolism
-
-
?
2,5-dihydroxypyridine + O2 + H2O
N-formylmaleamic acid
show the reaction diagram
-
activation with 10 mM DTT and 0.5 mM FeSO4 (25 min, 25C)
ring cleavage between carbon 5 and 6, further conversion to formic and maleamic acid is catalyzed by the NicD protein, a deformylase similar to some members of the alpha/beta-hydolase fold superfamily
-
?
additional information
?
-
-
aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation, no activity with: 2,3-dihydroxypyridine, 2,4-dihydroxypyridine, 2-hydroxypyridine, 3-hydroxypyridine, 4-hydroxypyridine, NA, 6HNA, 2-carboxypyridine, pyridoxamine, pyridoxal, catechol, protocatechuate, gentisate, gallate, resorcinol, hydroquinone, or pyrogallol
-
-
-
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
2,5-dihydroxypyridine + O2
N-formylmaleamic acid
show the reaction diagram
-
-
-
-
?
2,5-dihydroxypyridine + O2
N-formylmaleamic acid
show the reaction diagram
-
aerobic catabolism of nicotinic acid
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
nicotinic acid catabolism
-
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
-
nicotinic acid catabolism
-
-
?
2,5-dihydroxypyridine + O2 + H2O
N-formylmaleamic acid
show the reaction diagram
-
activation with 10 mM DTT and 0.5 mM FeSO4 (25 min, 25C)
ring cleavage between carbon 5 and 6, further conversion to formic and maleamic acid is catalyzed by the NicD protein, a deformylase similar to some members of the alpha/beta-hydolase fold superfamily
-
?
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
Pseudomonas fluorescens N-9
-
nicotinic acid catabolism
-
-
?
additional information
?
-
-
aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Fe2+
-
one mole per mole enzyme, activation of enzyme with 0.5 mM FeSO4 for 15 min at 25C, replacement with other divalent cations do not lead to detectable 2,5-dihydroxypyridine dioxygenase activity; purified enzyme is inactive, Fe2+-dependent reactivation of the purified enzyme (0.025 mM), 0.0198 mM iron is detected, indicating that the ratio of mol of iron to mol of a monomer of NicX is near 1.0
Fe2+
-
dependent on
Iron
-
enzyme contains loosly bound Fe3+; Fe2+ requirement
Iron
Gram-negative rod
-
Fe2+ requirement
Iron
-
Fe2+ requirement
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
-
-
2,2'-dipyridyl
Gram-negative rod
-
90% inhibition at 0.5 mM
EDTA
-
iron chelant
H2O2
-
oxidizing agent
iodoacetamide
-
-
N-ethylmaleimide
-
-
p-chloromercuribenzoate
-
-
Pyridine-1,2-diol
-
-
-
Pyridine-1,4-diol
-
-
-
pyridine-2,3-diol
-
-
pyridine-2,4-diol
-
-
Pyridine-3,4-diol
-
-
Tiron
-
i.e. 4,5-dihydroxy-1,3-benzene disulfonic acid
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
glutathione
Gram-negative rod
-
requirement
L-cysteine
-
enzyme has a specific requirement for L-cysteine (6.7 mM), required to restore full activity after dialysis or treatment with chelating agents
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.07
-
2,5-dihydroxypyridine
-
25C; pH 8.0, 25C, strict substrate specificity
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2.3
-
-
Vmax, pH 8.0, 25C
6
-
-
pH 8.0, 25C, in cell extracts of Escherichia coli BL21(DE3) overexpressing the nicX gene
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
-
Gram-negative rod
-
-
8
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
30
-
-
assay at
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
Pseudomonas fluorescens N-9
-
-
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
39000
-
-
SDS-PAGE
143200
-
-
gel filtration and mass spectrometry
242000
-
-
sucrose density gradient centrifugation (+ dithiothreitol)
330000
340000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 39500, SDS-PAGE
?
-
x * 39000, SDS-PAGE
hexamer
-
6 * 38 883, a hexameric assembly composed of two cyclic trimers, gel filtration and mass spectrometry
monomer
-
1 * 39000, amino acid sequence similarity to aminopeptidase S from Staphylococcus aureus and aminopeptidase T from Thermus thermophilus, 2 globular domains
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
purified recombinant NicX, hanging drop vapour diffusion method, 0.001 ml of 14 mg/ml protein in 20 mM HEPES pH 7.5, 0.1 M NaCl, and 2 mM DTT, is mixed with 0.001 ml of reservoir solution, containing 22% w/v PEG 4000, 0.1 M sodium acetate and 0.1 M HEPES pH 7.5, 2 mM DTT, and with 1 ml 50mM EDTA, 18C, method optimization, X-ray diffraction structure determination and analysis at 2.0 A resolution
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
60
-
-
10 min, enzyme in crude extract stable
65
-
-
10 min, enzyme in crude extract, 75% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
dialysis against phosphate buffer: complete loss of activity
-
incubation with or dialysis against 0.001 mM 8-hydroxyquinoline and by (NH4)2SO4 fractionation: complete loss of activity
-
L-cysteine: enzyme has a specific requirement for L-cysteine, 6.7 mM, required to restore full activity after dialysis or treatment with chelating agents
-
combination of dithiothreitol and FeSO4 is detrimental during long-term incubation at 0C
-
dithiothreitol stabilizes
-
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
rapid loss of activity in air, purified enzyme, t1/2 is 2 h
-
396336
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, stable for at least several weeks, enzyme in crude extract
-
4C, several days, enzyme in crude extract
-
-40C, 3 months, 20% loss of activity
-
0C, 20 mM sodium phosphate, pH 7.5, half-life: 2-3 days, addition of dithiothreitol extends half-life to about 2 weeks
-
4C, purified recombinant enzyme, 20 mM HEPES pH 7.5, 0.1 M NaCl, and 2 mM DTT, maintains its activity for several days
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
Escherichia coli cells overexpressing NicX are harvested and disrupted by passage through a French press, supernatant applied to DEAE-cellulose column with 50 mM NaH2PO4 buffer, pH 7.5, washed with buffer (0.1-0.3 M) containing 0.1 M NaCl, fraction with 2,5-dihydroxypyridine deoxygenase activity pooled and dialyzed with 20 mM NaH2PO4 buffer, pH 7.5, loaded onto hydroxyapatite column, eluted with 20-100 mM NaH2PO4 buffer, pH 7.0, fractions with enzyme activity pooled, dialyzed in 20 mM NaH2PO4 buffer, pH 7.5, and concentrated; recombinant enzyme
-
recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
functional overexpression in Escherichia coli strain BL21 (DE3)
-
PCR-amplification of nicX gene, overexpression of nix gene in Escherichia coli BL21(DE3) cells with plasmid pETNicX, for knockout mutants transformation of pKnicX plasmid into Pseudomonas putida KT2440 as recipient, using Escherichia coli DH10B(pKnicX) as donor strain, Escherichia coli HB101 (pRK600) as helper strain; the nicX gene is overexpressed in Escherichia coli BL21(DE3) cells containing plasmid pETNicX
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
nic gene cluster knockout mutants do not grow on nicotinic acid as sole carbon source in contrary to the wild-type, this ability can be restored by introducing a broad-host-range plasmid harboring a 14 kb DNA cassette containing the complete nice cluster, NicX knockout mutant does not show 2,5DHP dioxygenase activity
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
biotechnology
-
the enzyme catalyzes one step in a new process of detoxification/biotransformation of N-heterocyclic aromatic compounds