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Literature summary extracted from

  • 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 (2008), Proc. Natl. Acad. Sci. USA, 105, 11329-11334.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.13.11.9 biotechnology the enzyme catalyzes one step in a new process of detoxification/biotransformation of N-heterocyclic aromatic compounds Pseudomonas putida

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.9 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 Pseudomonas putida
1.13.11.9 the nicX gene is overexpressed in Escherichia coli BL21(DE3) cells containing plasmid pETNicX Pseudomonas putida
1.14.13.114 the nicC gene is cloned and expressed in Pseudomonas fluorescens (plasmid pIZNicC) Pseudomonas putida
3.5.1.106 expression in Escherichia coli Pseudomonas putida
3.5.1.107 overexpression in Escherichia coli BL21 (plasmid pETNicF) Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.9 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 Pseudomonas putida
3.5.1.106 D125A mutation leads to a complete loss of the deformylase activity Pseudomonas putida
3.5.1.106 E221A 70% of wild-type deformylase acticity Pseudomonas putida
3.5.1.106 H245A mutation leads to a complete loss of the deformylase activity Pseudomonas putida
3.5.1.106 S101A mutation leads to a complete loss of the deformylase activity Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.13.11.9 EDTA iron chelant Pseudomonas putida
1.13.11.9 H2O2 oxidizing agent Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.9 0.07
-
 2,5-dihydroxypyridine 25°C Pseudomonas putida
1.13.11.9 0.07
-
 2,5-dihydroxypyridine pH 8.0, 25°C, strict substrate specificity Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.9 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 Pseudomonas putida
1.13.11.9 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 Pseudomonas putida

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.13.11.9 39000
-
 SDS-PAGE Pseudomonas putida
1.14.13.114 42000
-
 x * 42000, SDS-PAGE Pseudomonas putida
1.17.2.1 12800
-
 x * 23800, small subunit NicA, + x * 12800, large subunit NicB Pseudomonas putida
1.17.2.1 23800
-
 x * 23800, small subunit NicA, + x * 12800, large subunit NicB Pseudomonas putida
3.5.1.106 29000
-
 x * 29000, SDS-PAGE Pseudomonas putida
3.5.1.106 29100
-
 x * 29100, calculated from sequence Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.9 2,5-dihydroxypyridine + O2 Pseudomonas putida activation with 10 mM DTT and 0.5 mM FeSO4 (25 min, 25°C) N-formylmaleamic acid 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 ?
1.13.11.9 2,5-dihydroxypyridine + O2 Pseudomonas putida aerobic catabolism of nicotinic acid N-formylmaleamic acid
-
 ?
1.13.11.9 2,5-dihydroxypyridine + O2 Pseudomonas putida KT 2240 activation with 10 mM DTT and 0.5 mM FeSO4 (25 min, 25°C) N-formylmaleamic acid 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 ?
1.13.11.9 2,5-dihydroxypyridine + O2 Pseudomonas putida KT 2240 aerobic catabolism of nicotinic acid N-formylmaleamic acid
-
 ?
1.13.11.9 additional information Pseudomonas putida aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation ?
-
 ?
1.13.11.9 additional information Pseudomonas putida KT 2240 aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation ?
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2 Pseudomonas putida
-
 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
3.5.1.106 N-formylmaleamic acid + H2O Pseudomonas putida aerobic catabolism of nicotinic acid maleamate + formate
-
 ?
3.5.1.107 maleamate + H2O Pseudomonas putida aerobic catabolism of nicotinic acid maleate + NH3
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.9 Pseudomonas putida
-
-
-
1.13.11.9 Pseudomonas putida
-
 KT2440
-
1.13.11.9 Pseudomonas putida KT 2240
-
-
-
1.13.11.9 Pseudomonas putida KT 2240
-
 KT2440
-
1.14.13.114 Pseudomonas putida
-
-
-
1.14.13.114 Pseudomonas putida KT 2240
-
-
-
1.17.2.1 Pseudomonas putida
-
 KT2440
-
1.17.2.1 Pseudomonas putida KT 2240
-
 KT2440
-
3.5.1.106 Pseudomonas putida
-
-
-
3.5.1.106 Pseudomonas putida KT 2240
-
-
-
3.5.1.107 Pseudomonas putida
-
-
-
3.5.1.107 Pseudomonas putida KT 2240
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.9 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 Pseudomonas putida
1.13.11.9 recombinant enzyme Pseudomonas putida

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.13.11.9 2.3
-
 Vmax, pH 8.0, 25°C Pseudomonas putida
1.13.11.9 6
-
 pH 8.0, 25°C, in cell extracts of Escherichia coli BL21(DE3) overexpressing the nicX gene Pseudomonas putida
3.5.1.107 0.0193
-
-
 Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.9 2,5-dihydroxypyridine + O2 activation with 10 mM DTT and 0.5 mM FeSO4 (25 min, 25°C) Pseudomonas putida N-formylmaleamic acid 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 ?
1.13.11.9 2,5-dihydroxypyridine + O2 aerobic catabolism of nicotinic acid Pseudomonas putida N-formylmaleamic acid
-
 ?
1.13.11.9 2,5-dihydroxypyridine + O2 extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6 Pseudomonas putida N-formylmaleamic acid
-
 ?
1.13.11.9 2,5-dihydroxypyridine + O2 activation with 10 mM DTT and 0.5 mM FeSO4 (25 min, 25°C) Pseudomonas putida KT 2240 N-formylmaleamic acid 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 ?
1.13.11.9 2,5-dihydroxypyridine + O2 aerobic catabolism of nicotinic acid Pseudomonas putida KT 2240 N-formylmaleamic acid
-
 ?
1.13.11.9 2,5-dihydroxypyridine + O2 extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6 Pseudomonas putida KT 2240 N-formylmaleamic acid
-
 ?
1.13.11.9 additional information aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation Pseudomonas putida ?
-
 ?
1.13.11.9 additional information 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 Pseudomonas putida ?
-
 ?
1.13.11.9 additional information aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation Pseudomonas putida KT 2240 ?
-
 ?
1.13.11.9 additional information 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 Pseudomonas putida KT 2240 ?
-
 ?
1.14.13.114 6-hydroxynicotinate + NADH + H+ + O2
-
 Pseudomonas putida 2,5-dihydroxypyridine + NAD+ + H2O + CO2
-
 ?
1.17.2.1 3-cyanopyridine + 5-methylphenazinium methyl sulfate + H2O 4% of the activity with nicotinate Pseudomonas putida ?
-
 ?
1.17.2.1 nicotinic acid + 5-methylphenazinium methyl sulfate + H2O
-
 Pseudomonas putida 6-hydroxynicotinic acid + ?
-
 ?
1.17.2.1 nipecotic acid + 5-methylphenazinium methyl sulfate + H2O 8% of the activity with nicotinate Pseudomonas putida ?
-
 ?
1.17.2.1 picolinic acid + 5-methylphenazinium methyl sulfate + H2O 7% of the activity with nicotinate Pseudomonas putida ?
-
 ?
1.17.2.1 pyrazine-2-carboxylic acid + 5-methylphenazinium methyl sulfate + H2O 8% of the activity with nicotinate Pseudomonas putida ?
-
 ?
1.17.2.1 pyridine-3-carboxyaldehyde + 5-methylphenazinium methyl sulfate + H2O 5% of the activity with nicotinate Pseudomonas putida ?
-
 ?
1.17.2.1 pyridine-3-sulfonic acid + 5-methylphenazinium methyl sulfate + H2O 4% of the activity with nicotinate Pseudomonas putida ?
-
 ?
3.5.1.106 N-formylmaleamic acid + H2O aerobic catabolism of nicotinic acid Pseudomonas putida maleamate + formate
-
 ?
3.5.1.106 N-formylmaleamic acid + H2O S101, D125, and H245 are essential for the enzyme activity, constituting the catalytic triad of the NicD deformylase Pseudomonas putida maleamate + formate
-
 ?
3.5.1.107 maleamate + H2O
-
 Pseudomonas putida maleate + NH3
-
 ?
3.5.1.107 maleamate + H2O aerobic catabolism of nicotinic acid Pseudomonas putida maleate + NH3
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.11.9 ? x * 39000, SDS-PAGE Pseudomonas putida
1.13.11.9 monomer 1 * 39000, amino acid sequence similarity to aminopeptidase S from Staphylococcus aureus and aminopeptidase T from Thermus thermophilus, 2 globular domains Pseudomonas putida
1.14.13.114 ? x * 42000, SDS-PAGE Pseudomonas putida
1.17.2.1 ? x * 23800, small subunit NicA, + x * 12800, large subunit NicB Pseudomonas putida
1.17.2.1 More small subunit NicA harbors the 41-C-X4-C-G-X-C-Xn-C-59 and 100-C-G-X-C-X31-C-X-C-137 conserved motifs likely involved in binding of the two [2Fe-2S] clusters Pseudomonas putida
1.17.2.1 More the N-terminal region of large subunit NicB contains the active site and the molybdopterin cytosine dinucleotide binding sites in an arrangement MPT2-MPT1-MPT3. The C-terminal region of NicB, residues750-1187, contains three conserved cytochrome c hemebinding motifs 817-CAVCH-823, 963-CTACH-969, and 1087-CLGCH-1093 Pseudomonas putida
3.5.1.106 ? x * 29000, SDS-PAGE Pseudomonas putida
3.5.1.106 ? x * 29100, calculated from sequence Pseudomonas putida

Synonyms

EC Number Synonyms Comment Organism
1.13.11.9 2,5-dihydroxypyridine dioxygenase
-
 Pseudomonas putida
1.13.11.9 2,5DHP dioxygenase
-
 Pseudomonas putida
1.13.11.9 NicX
-
 Pseudomonas putida
1.13.11.9 NicX protein
-
 Pseudomonas putida
1.14.13.114 6HNA monooxygenase
-
 Pseudomonas putida
1.14.13.114 NicC
-
 Pseudomonas putida
1.17.2.1 NicA
-
 Pseudomonas putida
1.17.2.1 NicB
-
 Pseudomonas putida
3.5.1.106 NicD
-
 Pseudomonas putida
3.5.1.107 NicF
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.9 25
-
-
 Pseudomonas putida
1.17.2.1 30
-
-
 Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.9 8
-
-
 Pseudomonas putida
1.17.2.1 7.5
-
-
 Pseudomonas putida

Cofactor

EC Number Cofactor Comment Organism Structure
1.13.11.9 heme
-
 Pseudomonas putida
1.14.13.114 NADH
-
 Pseudomonas putida
1.17.2.1 2Fe-2S-center
-
 Pseudomonas putida
1.17.2.1 cytochrome c
-
 Pseudomonas putida
1.17.2.1 molybdopterin cytosine dinucleotide
-
 Pseudomonas putida