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

Substrates: aerobic catabolism of nicotinic acid
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: a series of combined quantum mechanics and molecular mechanics (QM/MM) calculations is performed to illuminate the catalytic mechanism of 2,5-dihydroxypyridine dioxygenase (NicX). On the basis of the calculations, the most plausible reaction pathway and the structures of transition states and intermediates involved in the reaction, as well as the energy profiles, are described in detail
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: two key factors may influence the substrate specificity of the enzyme (NicX): 1. the successful binding of substrates in the active center, in which the pocket residues, including His189, His105, and Glu177, play a key role in substrate recognition, 2. the activation of the substrate by electron transfer from the substrate to dioxygen. Based on the crystal structure of the 2,5-dihydroxypyridine dioxygenase (NicX), an enzyme-substrate complex model is constructed and QM/MM calculations are carried out to unravel the mechanism of NicX-catalyzed heterocyclic ring cleavage involved in the biochemical degradation of nicotine
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: aerobic catabolism of nicotinic acid
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: -
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: -
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Gram-negative rod

Substrates: O2 cannot be replaced by methylene blue
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: -
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: nicotinic acid catabolism
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: -
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: nicotinic acid catabolism
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

396333

Substrates: -
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: strictly specific for 2,5-dihydroxypyridine
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: nicotinic acid catabolism
Products: -

additional information

Substrates: aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation
Products: -

additional information

Substrates: 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
Products: -

additional information

Substrates: aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation
Products: -

additional information

show all columns Go to Natural Substrate Search

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NATURAL SUBSTRATE

NATURAL PRODUCT

REACTION DIAGRAM

ORGANISM

UNIPROT

LITERATURE

COMMENTARY

REVERSIBILITY
r=reversible
ir=irreversible
?=not specified

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

10 entries

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

3 entries

additional information

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: -
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: -
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: -
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: -
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

764843, 765451

Substrates: -
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: aerobic catabolism of nicotinic acid
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: aerobic catabolism of nicotinic acid
Products: -

2,5-dihydroxypyridine + O2

N-formylmaleamic acid

Substrates: -
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: nicotinic acid catabolism
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: nicotinic acid catabolism
Products: -

2,5-dihydroxypyridine + O2 + H2O

maleamate + formate

Substrates: nicotinic acid catabolism
Products: -

additional information

Substrates: aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation
Products: -

additional information

Substrates: aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation
Products: -

show all columns Go to Cofactor Search

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COFACTOR

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

heme

show all columns Go to Metals and Ions Search

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METALS and IONS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

Fe2+

6 entries

Iron

4 entries

Fe2+

Fe2+-dependent enzyme

Fe2+

dependent on

Fe2+

one mole per mole enzyme, activation of enzyme with 0.5 mM FeSO4 for 15 min at 25°C, replacement with other divalent cations do not lead to detectable 2,5-dihydroxypyridine dioxygenase activity

Fe2+

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+

mononuclear non-heme iron enzyme

Fe2+

the enzyme uses a mononuclear non-heme iron enzyme to catalyze the oxidative pyridine ring cleavage reaction

765451

Iron

Fe2+ requirement

Iron

Gram-negative rod

Fe2+ requirement

Iron

Fe2+ requirement

Iron

enzyme contains loosly bound Fe3+

show all columns Go to Inhibitor Search

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INHIBITOR

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

1,10-phenanthroline

2,2'-dipyridyl

EDTA

H2O2

iodoacetamide

KCN

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

show all columns Go to Activating Compound Search

2,2'-dipyridyl

Gram-negative rod

90% inhibition at 0.5 mM

2,2'-dipyridyl

EDTA

EDTA

iron chelant

H2O2

H2O2

oxidizing agent

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ACTIVATING COMPOUND

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

DTT

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

show all columns Go to KM Value Search

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KM VALUE [mM]

SUBSTRATE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

IMAGE

0.07

2,5-dihydroxypyridine

0.07

2,5-dihydroxypyridine

25°C

0.07

2,5-dihydroxypyridine

pH 8.0, 25°C, strict substrate specificity

show all columns Go to Specific Activity Search

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SPECIFIC ACTIVITY [µmol/min/mg]

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

0.13

pyridine-2,6-dicarboxylic acid-induced cells of strain 23K8, pH 7.2, 30°C

15.3

2.3

Vmax, pH 8.0, 25°C

38.3

pH 8.0, 25°C, in cell extracts of Escherichia coli BL21(DE3) overexpressing the nicX gene

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pH OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

7.2

assay at

show all columns Go to Temperature Optimum Search

7.5

Gram-negative rod

assay at

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TEMPERATURE OPTIMUM

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

assay at

assay at

assay at

show all columns Go to Organism Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks include all textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks + FRENDAco-occurrence of enzyme names and organism names (less precise)

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ORGANISM

COMMENTARY

LITERATURE

UNIPROT

SEQUENCE DB

SOURCE

G2 and 2L

brenda

brenda

i.e. Wautersia campinensis

brenda

i.e. Wautersia campinensis

brenda

Gram-negative rod

brenda

brenda

3 entries

396333, 396334, 700940, 710759

brenda

764843, 765451

SwissProt

brenda

KT2440

brenda

700940, 710759

brenda

KT2440

brenda

show all columns Go to Source Tissue Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks

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SOURCE TISSUE

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

SOURCE

cell culture

cell culture

pyridine-2,6-dicarboxylic acid- and pyridine-2,3-dicarboxylic acid-induced strain 23K8 cells. The cells do not grow on pyridine-2,5-dicarboxylic acid

brenda

cell culture

pyridine-2,6-dicarboxylic acid- and pyridine-2,3-dicarboxylic acid-induced strain 23K8 cells. The cells do not grow on pyridine-2,5-dicarboxylic acid

brenda

show all columns Go to Localization Search

only manually annotated data include textmining data from AMENDAenzyme occurrence in organism, localization and source tissue with reliability ranks

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LOCALIZATION

ORGANISM

UNIPROT

COMMENTARY

GeneOntology No.

LITERATURE

SOURCE

cytosol

cytosol

5829

brenda

cytosol

5829

brenda

top hide

Highest Expressing Human Cell Lines

Filter by:

Cell Line Links

Gene Links

show all columns Go to General Information Search

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GENERAL INFORMATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

metabolism

physiological function

the enzyme is involved in the 3-hydroxypyridine degradation pathway

metabolism

the enzyme catalyzes the last step of putative pathway of pyridine-2,3-dicarboxylic acid oxidation in Cupriavidus campinensis 23K8

metabolism

the enzyme catalyzes the last step of putative pathway of pyridine-2,3-dicarboxylic acid oxidation in Cupriavidus campinensis 23K8

Go to AA Sequence and Transmembrane Helices SearchGo to Localization Prediction

Show AA Sequence and Transmembrane Helices (278 entries)
Please use the AA Sequence and Transmembrane Helices Search for a specific query.

Go to PDB and Structure Links Search

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PDB

SCOP

CATH

UNIPROT

ORGANISM

3 entries

7cn3, download, 3D-view

SCOPe (7cn3)

CATH (7cn3)

7cnt, download, 3D-view

SCOPe (7cnt)

CATH (7cnt)

7cup, download, 3D-view

SCOPe (7cup)

CATH (7cup)

show all columns Go to Molecular Weight Search

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MOLECULAR WEIGHT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

143200

gel filtration and mass spectrometry

242000

show all columns Go to Subunit Search

sucrose density gradient centrifugation (+ dithiothreitol)

330000 - 340000

gel filtration

39000

SDS-PAGE

39500

x * 39500, SDS-PAGE

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SUBUNIT

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

hexamer

monomer

x * 39000, SDS-PAGE

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

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

monomer

1 * 39000, amino acid sequence similarity to aminopeptidase S from Staphylococcus aureus and aminopeptidase T from Thermus thermophilus, 2 globular domains

Go to Crystallization (commentary) Search

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CRYSTALLIZATION (Commentary)

ORGANISM

UNIPROT

LITERATURE

396333, 396334

hanging-drop vapor-diffusion method at 20°C, crystal structure for the resting form of the enzyme (NicX), as well as a complex structure wherein 2,5-hydroxy-pyridine and N-formylmaleamic acid are trapped in different subunits. The resting state structure displays an octahedral coordination for Fe(II) with two histidine residues (His265 and His318), a serine residue (Ser302), a carboxylate ligand (Asp320), and two water molecules. 2,5-Hydroxy-pyridine does not bind as a ligand to Fe(II), yet its interactions with Leu104 and His105 function to guide and stabilize the substrate to the appropriate position to initiate the reaction

765451

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, 18°C, method optimization, X-ray diffraction structure determination and analysis at 2.0 A resolution

show all columns Go to Protein Variants Search

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PROTEIN VARIANTS

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

additional information

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

additional information

show all columns Go to Temperature Stability Search

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TEMPERATURE STABILITY

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

10 min, enzyme in crude extract stable

10 min, enzyme in crude extract, 75% loss of activity

Go to General Stability Search

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GENERAL STABILITY

ORGANISM

UNIPROT

LITERATURE

combination of dithiothreitol and FeSO4 is detrimental during long-term incubation at 0°C

396333, 396334

dialysis against phosphate buffer: complete loss of activity

dithiothreitol stabilizes

396333, 396334

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

Go to Oxidation Stability Search

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OXIDATION STABILITY

ORGANISM

UNIPROT

LITERATURE

rapid loss of activity in air, purified enzyme, t1/2 is 2 h

396336

Go to Storage Stability Search

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STORAGE STABILITY

ORGANISM

UNIPROT

LITERATURE

-20°C, stable for at least several weeks, enzyme in crude extract

-40°C, 3 months, 20% loss of activity

0°C, 20 mM sodium phosphate, pH 7.5, half-life: 2-3 days, addition of dithiothreitol extends half-life to about 2 weeks

4°C, purified recombinant enzyme, 20 mM HEPES pH 7.5, 0.1 M NaCl, and 2 mM DTT, maintains its activity for several days

4°C, 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

partial

recombinant enzyme

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)

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

phylogenetic analysis

the nicX gene is overexpressed in Escherichia coli BL21(DE3) cells containing plasmid pETNicX

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APPLICATION

ORGANISM

UNIPROT

COMMENTARY

LITERATURE

biotechnology

biotechnology

the enzyme catalyzes one step in a new process of detoxification/biotransformation of N-heterocyclic aromatic compounds

biotechnology

top print hide References Search

the enzyme catalyzes one step in a new process of detoxification/biotransformation of N-heterocyclic aromatic compounds

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REF.

AUTHORS

TITLE

JOURNAL

VOL.

PAGES

YEAR

ORGANISM (UNIPROT)

PUBMED ID

SOURCE

Behrman, E.J.; Stanier, R.Y.

The bacterial oxidation of nicotinic acid

J. Biol. Chem.

228

923-945

1957

Pseudomonas fluorescens

13475371

brenda

396333

Nozaki, M.

Nonheme iron dioxygenase

Mol. Mech. Oxygen Activ. (Hayaishi, O., ed.) Academic Press, New York

135-165

1974

Pseudomonas putida

brenda

Gauthier, J.J.; Rittenberg, S.C.

The metabolism of nicotinic acid. I. Purification and properties of 2,5-dihydroxypyridine oxygenase from Pseudomonas putida N-9

J. Biol. Chem.

246

3737-3742

1971

Pseudomonas putida

5578917

brenda

Orpin, C.G.; Knight, M.; Evans, W.C.

The bacterial oxidation of picolinamide, a photolytic product of Diquat

Biochem. J.

127

819-831

1972

Gram-negative rod

4342498

brenda

Cain, R.B.; Houghton, C.; Wright, K.A.

Microbial metabolism of the pyridine ring. Metabolism of 2- and 3-hydroxypyridines by the maleamate pathway in Achromobacter sp

Biochem. J.

140

293-300

1974

Achromobacter sp.

4455192

brenda

Jimenez, J.; Canales, A.; Jimenez-Barbero, J.; Ginalski, K.; Rychlewski, L.; Garcia, J.; Diaz, E.

Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440

Proc. Natl. Acad. Sci. USA

105

11329-11334

2008

Pseudomonas putida, Pseudomonas putida KT 2240

18678916

brenda

Jimenez, J.I.; Acebron, I.; Garcia, J.L.; Diaz, E.; Mancheno, J.M.

A preliminary crystallographic study of recombinant NicX, an Fe2+-dependent 2,5-dihydroxypyridine dioxygenase from Pseudomonas putida KT2440

Acta Crystallogr. Sect. F

549-553

2010

Pseudomonas putida, Pseudomonas putida KT 2240

20445257

brenda

Kutanovas, S.; Karvelis, L.; Vaitekunas, J.; Stankeviciute, J.; Gasparaviciute, R.; Meskys, R.

Isolation and characterization of novel pyridine dicarboxylic acid-degrading microorganisms

Chemija

74-83

2016

Cupriavidus campinensis, Cupriavidus campinensis 23K8

brenda

Zhao, S.; Hu, C.; Guo, L.; Li, K.; Yu, H.

Isolation of a 3-hydroxypyridine degrading bacterium, Agrobacterium sp. DW-1, and its proposed degradation pathway

AMB Express

2019

Agrobacterium sp. DW-1

31102032

brenda