Literature summary for 1.13.11.9 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.

Search for more literature for 1.13.11.9

Application

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

Cloned(Commentary)

Cloned (Comment)	Organism
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
the nicX gene is overexpressed in Escherichia coli BL21(DE3) cells containing plasmid pETNicX	Pseudomonas putida

Protein Variants

Protein Variants	Comment	Organism
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

Inhibitors

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

KM Value [mM]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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
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]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
39000	-	SDS-PAGE	Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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	?
2,5-dihydroxypyridine + O2	Pseudomonas putida	aerobic catabolism of nicotinic acid	N-formylmaleamic acid	-	?
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	?
2,5-dihydroxypyridine + O2	Pseudomonas putida KT 2240	aerobic catabolism of nicotinic acid	N-formylmaleamic acid	-	?
additional information	Pseudomonas putida	aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation	?	-	?
additional information	Pseudomonas putida KT 2240	aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	-	-	-
Pseudomonas putida	-	KT2440	-
Pseudomonas putida KT 2240	-	-	-
Pseudomonas putida KT 2240	-	KT2440	-

Purification (Commentary)

Purification (Comment)	Organism
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
recombinant enzyme	Pseudomonas putida

Purification (Comment)

Organism

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

recombinant enzyme

Pseudomonas putida

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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	?
2,5-dihydroxypyridine + O2	aerobic catabolism of nicotinic acid	Pseudomonas putida	N-formylmaleamic acid	-	?
2,5-dihydroxypyridine + O2	extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6	Pseudomonas putida	N-formylmaleamic acid	-	?
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	?
2,5-dihydroxypyridine + O2	aerobic catabolism of nicotinic acid	Pseudomonas putida KT 2240	N-formylmaleamic acid	-	?
2,5-dihydroxypyridine + O2	extradiol ring-cleavage dioxygenase cleaves between carbons 5 and 6	Pseudomonas putida KT 2240	N-formylmaleamic acid	-	?
additional information	aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation	Pseudomonas putida	?	-	?
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	?	-	?
additional information	aerobic nicotinic acid degradation, nice gene cluster is responsible for the aerobic nicotinic acid degradation	Pseudomonas putida KT 2240	?	-	?
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	?	-	?

Subunits

Subunits	Comment	Organism
?	x * 39000, SDS-PAGE	Pseudomonas putida
monomer	1 * 39000, amino acid sequence similarity to aminopeptidase S from Staphylococcus aureus and aminopeptidase T from Thermus thermophilus, 2 globular domains	Pseudomonas putida

Synonyms

Synonyms	Comment	Organism
2,5-dihydroxypyridine dioxygenase	-	Pseudomonas putida
2,5DHP dioxygenase	-	Pseudomonas putida
NicX	-	Pseudomonas putida
NicX protein	-	Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	-	Pseudomonas putida

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	-	Pseudomonas putida

Cofactor

Cofactor	Comment	Organism	Structure
heme	-	Pseudomonas putida