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Literature summary for 1.14.13.24 extracted from

  • Gao, X.; Tan, C.L.; Yeo, C.C.; Poh, C.L.
    Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867 (2005), J. Bacteriol., 187, 7696-7702.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
additional information isozyme II is strictly inducible by specific aromatic substrates Pseudomonas alcaligenes

Cloned(Commentary)

Cloned (Comment) Organism
gene xlnD, DNA and amino acid sequence determination and analysis, subcloning and overexpression of the His-tagged enzyme in Escherichia coli strain DH5alpha, S17-1, and BL21(DE3) Pseudomonas alcaligenes

Protein Variants

Protein Variants Comment Organism
additional information construction of a P25X xlnD knockout mutant strain G50, no induction after growth on lactate and 3-hydroxy-4-methylbenzoate in contrast to the wild-type enzyme, slightly reduced activity with 3-hydroxybenzoate and gentisate Pseudomonas alcaligenes

Inhibitors

Inhibitors Comment Organism Structure
Ca2+
-
Pseudomonas alcaligenes
Cu2+ complete inhibition at 0.05 mM Pseudomonas alcaligenes
Fe2+ complete inhibition at 5 mM Pseudomonas alcaligenes
Hg2+ complete inhibition at 0.05 mM Pseudomonas alcaligenes
Mn2+ complete inhibition at 5 mM Pseudomonas alcaligenes
Zn2+
-
Pseudomonas alcaligenes

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.071
-
3-hydroxy-4-methylbenzoate pH 7.5, 23°C, recombinant enzyme, cofactor NADPH Pseudomonas alcaligenes
0.079
-
3-hydroxybenzoate pH 7.5, 23°C, recombinant enzyme, cofactor NADH Pseudomonas alcaligenes
0.095
-
3-hydroxy-4-methylbenzoate pH 7.5, 23°C, recombinant enzyme, cofactor NADH Pseudomonas alcaligenes
0.108
-
3-hydroxybenzoate pH 7.5, 23°C, recombinant enzyme, cofactor NADPH Pseudomonas alcaligenes

Metals/Ions

Metals/Ions Comment Organism Structure
K+ slight activation of 13% at 5 mM Pseudomonas alcaligenes

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
43000
-
3 * 43000, His-tagged recombinant enzyme, SDS-PAGE Pseudomonas alcaligenes
130000
-
His-tagged recombinant enzyme, native PAGE and gel filtration Pseudomonas alcaligenes

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3-hydroxybenzoate + NAD(P)H + O2 Pseudomonas alcaligenes the enzyme is involved in the degradation of 2,5-xylenol and 3,5-xylenol via the gentisate pathway, overview 2,5-dihydroxybenzoate + NAD(P)+ + H2O
-
?
3-hydroxybenzoate + NAD(P)H + O2 Pseudomonas alcaligenes P25X the enzyme is involved in the degradation of 2,5-xylenol and 3,5-xylenol via the gentisate pathway, overview 2,5-dihydroxybenzoate + NAD(P)+ + H2O
-
?
additional information Pseudomonas alcaligenes degradation of 2,5-xylenol and 3,5-xylenol in strain P25X depends on isozyme I, not isozyme II ?
-
?
additional information Pseudomonas alcaligenes P25X degradation of 2,5-xylenol and 3,5-xylenol in strain P25X depends on isozyme I, not isozyme II ?
-
?

Organism

Organism UniProt Comment Textmining
Pseudomonas alcaligenes Q9F131 gene xlnD, two isozymes I and II
-
Pseudomonas alcaligenes P25X Q9F131 gene xlnD, two isozymes I and II
-

Purification (Commentary)

Purification (Comment) Organism
recombinnat His-tagged enzyme from Escherichia coli by metal chelating chromatography Pseudomonas alcaligenes

Source Tissue

Source Tissue Comment Organism Textmining
additional information effects of carbon sources for growth on enzyme substrate specificity, overview Pseudomonas alcaligenes
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.63
-
recombinant His-tagged enzyme, substrate 3-hydroxy-5-methylbenzoate Pseudomonas alcaligenes

Storage Stability

Storage Stability Organism
-80°C, purified recombinant enzyme, 50 mM MOPS or 100 mM potassium phosphate, 10% v/v glycerol, 0.2 mM FAD, 60% activity remaining after 3 months Pseudomonas alcaligenes
22°C, rom temperature, purified recombinant enzyme, 50 mM MOPS or 100 mM potassium phosphate, 10% v/v glycerol, 0.2 mM FAD, complete loss of activity after 12 h Pseudomonas alcaligenes
4°C, purified recombinant enzyme, 50 mM MOPS or 100 mM potassium phosphate, 10% v/v glycerol, 0.2 mM FAD, complete loss of activity after 3 days Pseudomonas alcaligenes

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3-aminobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes ?
-
?
3-aminobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes P25X ?
-
?
3-bromobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes ?
-
?
3-bromobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes P25X ?
-
?
3-chlorobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes ?
-
?
3-fluorobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes ?
-
?
3-hydroxy-3-methylbenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes 2,5-dihydroxy-3-methylbenzoate + NAD(P)+ + H2O
-
?
3-hydroxy-4-methylbenzoate + NAD(P)H + O2 the substrate is an intermediate in the degradation of 2,5-xylenol, higher activity with the recombinant His-tagged enzyme compared to the native enzyme Pseudomonas alcaligenes 2,5-dihydroxy-4-methylbenzoate + NAD(P)+ + H2O
-
?
3-hydroxy-5-methylbenzoate + NAD(P)H + O2 the substrate is an intermediate in the degradation of 3,5-xylenol, higher activity with the recombinant His-tagged enzyme compared to the native enzyme, preferred substrate Pseudomonas alcaligenes 2,5-dihydroxy-3-methylbenzoate + NAD(P)+ + H2O
-
?
3-hydroxybenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes 2,5-dihydroxybenzoate + NAD(P)+ + H2O i.e. gentisate ?
3-hydroxybenzoate + NAD(P)H + O2 the enzyme is involved in the degradation of 2,5-xylenol and 3,5-xylenol via the gentisate pathway, overview Pseudomonas alcaligenes 2,5-dihydroxybenzoate + NAD(P)+ + H2O
-
?
3-hydroxybenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes P25X 2,5-dihydroxybenzoate + NAD(P)+ + H2O i.e. gentisate ?
3-hydroxybenzoate + NAD(P)H + O2 the enzyme is involved in the degradation of 2,5-xylenol and 3,5-xylenol via the gentisate pathway, overview Pseudomonas alcaligenes P25X 2,5-dihydroxybenzoate + NAD(P)+ + H2O
-
?
3-methylthiobenzoate + NAD(P)H + O2
-
Pseudomonas alcaligenes ?
-
?
additional information degradation of 2,5-xylenol and 3,5-xylenol in strain P25X depends on isozyme I, not isozyme II Pseudomonas alcaligenes ?
-
?
additional information degradation of 2,5-xylenol and 3,5-xylenol in strain P25X depends on isozyme I, not isozyme II Pseudomonas alcaligenes P25X ?
-
?

Subunits

Subunits Comment Organism
trimer 3 * 43000, His-tagged recombinant enzyme, SDS-PAGE Pseudomonas alcaligenes

Synonyms

Synonyms Comment Organism
3-hydroxybenzoate-6-hydroxylase
-
Pseudomonas alcaligenes
XlnD
-
Pseudomonas alcaligenes

Temperature Optimum [°C]

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

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
15 50 no activity below 15°C and above 50°C Pseudomonas alcaligenes

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
-
Pseudomonas alcaligenes

pH Range

pH Minimum pH Maximum Comment Organism
6 9
-
Pseudomonas alcaligenes

Cofactor

Cofactor Comment Organism Structure
NADH XlnD can utilize either NADH or NADPH as the electron donor, NADH is preferred Pseudomonas alcaligenes
NADPH XlnD can utilize either NADH or NADPH as the electron donor, NADH is preferred Pseudomonas alcaligenes