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Literature summary for 1.13.11.4 extracted from

  • Feng, Y.; Khoo, H.E.; Poh, C.L.
    Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869 (1999), Appl. Environ. Microbiol., 65, 946-950.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
2-mercaptoethanol stimulated by 2 mM Pseudomonas putida
2-mercaptoethanol stimulated by 2 mM Pseudomonas alcaligenes
Fe2+
-
Pseudomonas putida
Fe2+
-
Pseudomonas alcaligenes
glycerol stimulated by 10% (v/v) Pseudomonas putida
glycerol stimulated by 10% (v/v) Pseudomonas alcaligenes
L-cysteine stimulated by 2 mM Pseudomonas putida
L-cysteine stimulated by 2 mM Pseudomonas alcaligenes

General Stability

General Stability Organism
highly unstable in 50 mM MOPS buffer with no additives Pseudomonas putida
highly unstable in 50 mM MOPS buffer with no additives Pseudomonas alcaligenes

Inhibitors

Inhibitors Comment Organism Structure
Cu2+ 5 mM complete inactivation; complete inhibition at 5 mM Pseudomonas alcaligenes
Cu2+ 5 mM complete inactivation; complete inhibition at 5 mM Pseudomonas putida
EDTA 60.24% residual activity at 5 mM Pseudomonas alcaligenes
EDTA 4.95% residual activity at 5 mM Pseudomonas putida
Mg2+ 66% residual activity at 5 mM Pseudomonas putida
Mn2+ 73.96% residual activity at 5 mM Pseudomonas alcaligenes
Mn2+ 22.36% residual activity at 5 mM Pseudomonas putida
additional information K+, Na+, Mg2+, and Ca2+ do not inhibit the activity Pseudomonas alcaligenes
additional information K+, Na+, and Ca2+, do not inhibit the activity Pseudomonas putida
Zn2+ 2.58% residual activity at 5 mM Pseudomonas alcaligenes
Zn2+ 4.69% residual activity at 5 mM Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0043
-
3-Isopropylgentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas putida
0.011
-
3-Bromogentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas putida
0.015
-
3-Bromogentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas alcaligenes
0.036
-
3-Methylgentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas putida
0.053
-
3-Isopropylgentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas alcaligenes
0.076
-
3-Methylgentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas alcaligenes
0.092
-
gentisate
-
Pseudomonas alcaligenes
0.092
-
gentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas alcaligenes
0.143
-
gentisate
-
Pseudomonas putida
0.143
-
gentisate in 0.1 M phosphate buffer, pH 7.4, at 23°C Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
Fe2+ strongly stimulated by 0.1 mM Fe2+ Pseudomonas putida
Fe2+ strongly stimulated by 0.1 mM Fe2+ Pseudomonas alcaligenes
additional information K+, Na+, and Ca2+, do not stimulate the activity Pseudomonas putida

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
39000
-
4 * 39000, SDS-PAGE Pseudomonas alcaligenes
41000
-
2 * 41000 Pseudomonas putida
41000
-
2 * 41000, gel filtration Pseudomonas putida
82000
-
gel filtration Pseudomonas putida
154000
-
gel filtration Pseudomonas alcaligenes

Organism

Organism UniProt Comment Textmining
Pseudomonas alcaligenes
-
-
-
Pseudomonas alcaligenes NCIB 9867
-
-
-
Pseudomonas putida
-
-
-
Pseudomonas putida NCIB 9869
-
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Pseudomonas putida
-
Pseudomonas alcaligenes
ammonium sulfate fractionation, DEAE-Sepharose fast flow column chromatography Pseudomonas putida
ammonium sulfate fractionation, DEAE-Sepharose fast flow column chromatography Pseudomonas alcaligenes

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.45
-
crude extract, at 23°C Pseudomonas alcaligenes
0.72
-
crude extract, at 23°C Pseudomonas putida
23.2
-
after 51.56fold purification, at 23°C Pseudomonas alcaligenes
43.14
-
after 59.92fold purification, at 23°C Pseudomonas putida

Storage Stability

Storage Stability Organism
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 12 months, 3.5% residual activity Pseudomonas putida
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 12 months, 42.9% residual activity Pseudomonas alcaligenes
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 3 months, 42.5% residual activity Pseudomonas putida
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 3 months, 86.5% residual activity Pseudomonas alcaligenes
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 6 months, 23.1% residual activity Pseudomonas putida
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 6 months, 70.9% residual activity Pseudomonas alcaligenes
4°C, MOPS buffer with no additives, 24 h, 22.5% residual activity Pseudomonas alcaligenes
4°C, MOPS buffer with no additives, 24 h, 33.9% residual activity Pseudomonas putida
4°C, MOPS buffer with no additives, 96 h, complete loss of activity Pseudomonas putida
4°C, MOPS buffer with no additives, 96 h, complete loss of activity Pseudomonas alcaligenes

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,5-dihydroxybenzoate + O2
-
Pseudomonas putida maleylpyruvate
-
?
2,5-dihydroxybenzoate + O2
-
Pseudomonas alcaligenes maleylpyruvate
-
?
2,5-dihydroxybenzoate + O2
-
Pseudomonas alcaligenes NCIB 9867 maleylpyruvate
-
?
2,5-dihydroxybenzoate + O2
-
Pseudomonas putida NCIB 9869 maleylpyruvate
-
?
3-bromogentisate + O2
-
Pseudomonas putida 3-bromo-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
-
?
3-bromogentisate + O2
-
Pseudomonas alcaligenes 3-bromo-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
-
?
3-bromogentisate + O2
-
Pseudomonas alcaligenes NCIB 9867 3-bromo-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
-
?
3-bromogentisate + O2
-
Pseudomonas putida NCIB 9869 3-bromo-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
-
?
3-isopropyl-gentisate + O2
-
Pseudomonas putida 4-hydroxy-3-isopropyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-isopropyl-gentisate + O2
-
Pseudomonas alcaligenes 4-hydroxy-3-isopropyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-isopropyl-gentisate + O2
-
Pseudomonas alcaligenes NCIB 9867 4-hydroxy-3-isopropyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-isopropyl-gentisate + O2
-
Pseudomonas putida NCIB 9869 4-hydroxy-3-isopropyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-methylgentisate + O2
-
Pseudomonas putida 4-hydroxy-3-methyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-methylgentisate + O2
-
Pseudomonas alcaligenes 4-hydroxy-3-methyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-methylgentisate + O2
-
Pseudomonas alcaligenes NCIB 9867 4-hydroxy-3-methyl-6-oxo-hepta-2,4-dienedioic acid
-
?
3-methylgentisate + O2
-
Pseudomonas putida NCIB 9869 4-hydroxy-3-methyl-6-oxo-hepta-2,4-dienedioic acid
-
?
gentisate + O2
-
Pseudomonas putida maleylpyruvate
-
?
gentisate + O2
-
Pseudomonas alcaligenes maleylpyruvate
-
?
gentisate + O2
-
Pseudomonas alcaligenes NCIB 9867 maleylpyruvate
-
?
gentisate + O2
-
Pseudomonas putida NCIB 9869 maleylpyruvate
-
?

Subunits

Subunits Comment Organism
dimer 2 * 41000 Pseudomonas putida
homodimer 2 * 41000, gel filtration Pseudomonas putida
homotetramer 4 * about 39000, gel filtration Pseudomonas alcaligenes
tetramer 4 * 39000, SDS-PAGE Pseudomonas alcaligenes

Synonyms

Synonyms Comment Organism
P25X gentisate 1,2-dioxygenase
-
Pseudomonas alcaligenes
P35X gentisate 1,2-dioxygenase
-
Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
-
Pseudomonas putida
50
-
-
Pseudomonas alcaligenes

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50
-
retains 35% of its activity when incubated at 50°C for 5 min Pseudomonas putida
50
-
retains 73% of its activity when incubated at 50°C for 5 min Pseudomonas alcaligenes

pH Optimum

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

pH Stability

pH Stability pH Stability Maximum Comment Organism
5 7.5
-
Pseudomonas alcaligenes
7 9
-
Pseudomonas putida
7 9
-
Pseudomonas alcaligenes

pI Value

Organism Comment pI Value Maximum pI Value
Pseudomonas putida isoelectric focusing 4.8 4.6
Pseudomonas alcaligenes isoelectric focusing
-
4.85