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 hide
1.13.11.9
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RECOMMENDED NAME
GeneOntology No.
2,5-dihydroxypyridine 5,6-dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2,5-dihydroxypyridine + O2 = N-formylmaleamic acid
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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redox reaction
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reduction
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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 I
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nicotine degradation II (pyrrolidine pathway)
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nicotine degradation III (VPP pathway)
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SYSTEMATIC NAME
IUBMB Comments
2,5-dihydroxypyridine:oxygen 5,6-oxidoreductase
Requires Fe2+.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-57-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
G2 and 2L
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Manually annotated by BRENDA team
Gram-negative rod
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,5-dihydroxypyridine + O2
N-formylmaleamic acid
show the reaction diagram
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
2,5-dihydroxypyridine + O2 + H2O
N-formylmaleamic acid
show the reaction diagram
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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
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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
2,5-dihydroxypyridine + O2
N-formylmaleamic acid
show the reaction diagram
2,5-dihydroxypyridine + O2 + H2O
maleamate + formate
show the reaction diagram
2,5-dihydroxypyridine + O2 + H2O
N-formylmaleamic acid
show the reaction diagram
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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
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?
additional information
?
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aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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2,2'-dipyridyl
iodoacetamide
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N-ethylmaleimide
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p-chloromercuribenzoate
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Pyridine-1,2-diol
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Pyridine-1,4-diol
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pyridine-2,3-diol
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pyridine-2,4-diol
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Pyridine-3,4-diol
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Tiron
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i.e. 4,5-dihydroxy-1,3-benzene disulfonic acid
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
glutathione
Gram-negative rod
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requirement
L-cysteine
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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]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.07
2,5-dihydroxypyridine
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25C; pH 8.0, 25C, strict substrate specificity
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.3
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Vmax, pH 8.0, 25C
6
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pH 8.0, 25C, in cell extracts of Escherichia coli BL21(DE3) overexpressing the nicX gene
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
Gram-negative rod
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39000
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SDS-PAGE
143200
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gel filtration and mass spectrometry
242000
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sucrose density gradient centrifugation (+ dithiothreitol)
330000 - 340000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
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6 * 38 883, a hexameric assembly composed of two cyclic trimers, gel filtration and mass spectrometry
monomer
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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
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 50 mM EDTA, 18C, method optimization, X-ray diffraction structure determination and analysis at 2.0 A resolution
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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10 min, enzyme in crude extract stable
65
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10 min, enzyme in crude extract, 75% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
combination of dithiothreitol and FeSO4 is detrimental during long-term incubation at 0C
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dialysis against phosphate buffer: complete loss of activity
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dithiothreitol stabilizes
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incubation with or dialysis against 0.001 mM 8-hydroxyquinoline and by (NH4)2SO4 fractionation: complete loss of activity
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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
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
rapid loss of activity in air, purified enzyme, t1/2 is 2 h
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396336
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, stable for at least several weeks, enzyme in crude extract
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-40C, 3 months, 20% loss of activity
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0C, 20 mM sodium phosphate, pH 7.5, half-life: 2-3 days, addition of dithiothreitol extends half-life to about 2 weeks
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4C, purified recombinant enzyme, 20 mM HEPES pH 7.5, 0.1 M NaCl, and 2 mM DTT, maintains its activity for several days
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4C, several days, enzyme in crude extract
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Purification/COMMENTARY
ORGANISM
UNIPROT
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
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recombinant enzyme from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
functional overexpression in Escherichia coli strain BL21 (DE3)
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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
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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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
COMMENTARY hide
LITERATURE
biotechnology
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the enzyme catalyzes one step in a new process of detoxification/biotransformation of N-heterocyclic aromatic compounds