Information on EC 1.14.13.24 - 3-hydroxybenzoate 6-monooxygenase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.14.13.24
-
RECOMMENDED NAME
GeneOntology No.
3-hydroxybenzoate 6-monooxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
3-hydroxybenzoate + NADH + H+ + O2 = 2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
-
-
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
2,5-xylenol and 3,5-xylenol degradation
-
-
3-chlorobenzoate degradation III (via gentisate)
-
-
m-cresol degradation
-
-
3-phenylpropionate degradation
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Benzoate degradation
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
3-hydroxybenzoate,NADH:oxygen oxidoreductase (6-hydroxylating)
A flavoprotein (FAD). Acts also on a number of analogues of 3-hydroxybenzoate substituted in the 2, 4, 5 and 6 positions; NADPH can act instead of NADH, but more slowly.
CAS REGISTRY NUMBER
COMMENTARY hide
51570-26-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene ncgl2923
UniProt
Manually annotated by BRENDA team
gene ncgl2923
UniProt
Manually annotated by BRENDA team
gene mhbM
SwissProt
Manually annotated by BRENDA team
orf2, gene nagX
-
-
Manually annotated by BRENDA team
orf2, gene nagX
-
-
Manually annotated by BRENDA team
gene xlnD, two isozymes I and II
SwissProt
Manually annotated by BRENDA team
gene xlnD, two isozymes I and II
SwissProt
Manually annotated by BRENDA team
gene narX
UniProt
Manually annotated by BRENDA team
gene narX
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,3,4-trihydroxybenzoate + NADH + H+ + O2
2,3,4,6-tetrahydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
2,3-dihydroxybenzoate + NADH + H+ + O2
2,3,6-trihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
2,3-dihydroxybenzoate + NADH + O2
2,3,5-trihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3,4-dihydroxybenzoate + NADH + H+ + O2
?
show the reaction diagram
-
very poor substrate
-
-
?
3,4-dihydroxybenzoate + NADH + O2
2,4,5-trihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3,5-dihydroxybenzoate + NADH + H+ + O2
2,3,5,6-tetrahydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
3,5-dihydroxybenzoate + NADH + O2
2,3,5-trihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3-aminobenzoate + NAD(P)H + O2
?
show the reaction diagram
3-bromobenzoate + NAD(P)H + O2
?
show the reaction diagram
3-chlorobenzoate + NAD(P)H + O2
?
show the reaction diagram
-
-
-
?
3-fluorobenzoate + NAD(P)H + O2
?
show the reaction diagram
-
-
-
?
3-hydroxy-3-methylbenzoate + NAD(P)H + O2
2,5-dihydroxy-3-methylbenzoate + NAD(P)+ + H2O
show the reaction diagram
-
-
-
?
3-hydroxy-4-aminobenzoate + NADH + H+ + O2
4-amino-2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
3-hydroxy-4-methoxybenzoate + NADH + H+ + O2
4-methoxy-2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
3-hydroxy-4-methylbenzoate + NAD(P)H + O2
2,5-dihydroxy-4-methylbenzoate + NAD(P)+ + H2O
show the reaction diagram
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
-
-
?
3-hydroxy-4-methylbenzoate + NADH + H+ + O2
4-methyl-2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
-
-
-
?
3-hydroxy-5-methylbenzoate + NAD(P)H + O2
2,5-dihydroxy-3-methylbenzoate + NAD(P)+ + H2O
show the reaction diagram
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
-
-
?
3-hydroxy-5-methylbenzoate + NADH + O2
2,5-dihydroxy-3-methylbenzoate + NAD+ + H2O
show the reaction diagram
3-hydroxybenzoate + NAD(P)H + H+ + O2
gentisate + NAD(P)+ + H2O
show the reaction diagram
3-hydroxybenzoate + NAD(P)H + O2
2,5-dihydroxybenzoate + NAD(P)+ + H2O
show the reaction diagram
3-hydroxybenzoate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3-hydroxybenzoate + NADH + O2
2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3-hydroxybenzoate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
show the reaction diagram
3-methylthiobenzoate + NAD(P)H + O2
?
show the reaction diagram
-
-
-
?
4-fluoro-3-hydroxybenzoate + NADH + O2
4-fluoro-2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
-
36% of the reaction with 3-hydroxybenzoate
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?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
3-hydroxybenzoate + NAD(P)H + H+ + O2
gentisate + NAD(P)+ + H2O
show the reaction diagram
3-hydroxybenzoate + NAD(P)H + O2
2,5-dihydroxybenzoate + NAD(P)+ + H2O
show the reaction diagram
3-hydroxybenzoate + NADH + H+ + O2
2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3-hydroxybenzoate + NADH + O2
2,5-dihydroxybenzoate + NAD+ + H2O
show the reaction diagram
3-hydroxybenzoate + NADPH + H+ + O2
2,5-dihydroxybenzoate + NADP+ + H2O
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
additional information
-
the enzyme harbors a phospholipid ligand. The purified enzyme obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of the cytoplasmic membrane of Escherichia coli. The enzyme that is produced in the host Rhodococcus jostii RHA#2 by employing a newly developed actinomycete expression system, contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. The lipid cofactor stabilizes monomer-monomer contact
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
slight activation of 13% at 5 mM
additional information
-
no requirement for metal ions
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cibacron blue
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50% inhibition at 0.04 mM
Diethylpyrocarbonate
Mn2+
complete inhibition at 5 mM
N-bromosuccinimide
p-chloromercuribenzoate
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at low concentrations
Phenylglyoxal
Urea
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70% loss of activity at 1 M
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,5-Dihydroxybenzoate
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i.e. gentisate, strongly stimulates the NADH oxidase activity of the enzyme
3-hydroxybenzoate
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strongly stimulates activity
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.051
2,3-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
0.14
2,5-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
0.25
3,5-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
0.071 - 0.095
3-hydroxy-4-methylbenzoate
0.027 - 0.19
3-hydroxybenzoate
0.04 - 2.5
NADH
0.064 - 0.86
NADPH
additional information
additional information
-
Km at various pH values
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10
2,3-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
7
2,5-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
13
3,5-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
0.0178 - 41
3-hydroxybenzoate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
200
2,3-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
50
2,5-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
50
3,5-Dihydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
800
3-hydroxybenzoate
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in 50 mM Tris-SO4 (pH 8.0), at 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.53
recombinant wild-type enzyme
1.63
recombinant His-tagged enzyme, substrate 3-hydroxy-5-methylbenzoate
5.8
purified recombinant His-tagged NarX, pH 7.4, 30C
6
-
crude extract, in 50 mM Tris-SO4 (pH 8.0), at 25C
6.92
purified recombinant enzyme, with NADH, pH 7.5, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
soluble enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8
-
at pH 5.5-8, the residual activity of soluble enzyme is less than that of the immobilized one
6 - 8
high activity range
6.5 - 11
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no activity below pH 6.5 and above pH 11
9 - 10
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at pH 9 and 10, the enzyme activity of of the soluble enzyme is higher than that of the immobilized enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10 - 50
-
inactive above 50C
15 - 40
-
no activity below 15C and above 40C
15 - 50
no activity below 15C and above 50C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40000 - 45000
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gel filtration, SDS-PAGE
41500
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gel filtration
42000
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1 * 42000, SDS-PAGE
45000
3 * 45000, recombinant His-tagged NarX, SDS-PAGE
49000
3 * 49000, recombinant Ncgl2923, SDS-PAGE
49900
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x * 49900, calculated from amino acid sequence
67000
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SDS-PAGE
70000
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gel filtration
85000
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sedimentation equilibrium measurement
108000
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gel filtration
119000
recombinant His-tagged NarX, gel filtration
125000
recombinant Ncgl2923, gel filtration
130000
His-tagged recombinant enzyme, native PAGE and gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
trimer
additional information
Ser166 and Arg169 form the highly conserved SXXR motif important for structure and function of the enzyme
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, using 28% (w/v) PEG 4000, 0.2 M sodium acetate, and 0.1 M Tris-HCl (pH 8.5)
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.5
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in phosphate and Tris-HCl buffer
438958
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
complete inactivation at room temperature without glycerol after 18 h
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immobilized onto surface-functionalized electrospun polycaprolactone (PCL) fibers can tolerate the changes in temperature and pH better than the free enzyme can. The immobilized enzyme can be reused at least 10 times with more than 60% of the original activity at pH 8 and 25C. The 3-hydroxybenzoate 6-hydroxylase fibers are potentially useful as a heterogeneous catalyst under the conditions in which free enzyme can not endure.
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80C, 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
22C, 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
4C, optimal storage in 50 mM phosphate or Tris buffer pH 8 with 10% glycerol
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4C, 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
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
98% homogeneity
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DEAE-Sepharose column chromatography and Sephadex G-25 gel filtration
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homogeneity
Ni-NTA agarose column chromatography and Q-Sepharose column chromatography
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recombinant His-tagged NarX from Escherichia coli strain Rosetta (DE3) pLysS by nickel affinity chromatography to homogeneity
recombinant N-terminally His-tagged Ncgl2923 from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity
recombinnat His-tagged enzyme from Escherichia coli by metal chelating chromatography
single step purification using substrate-mediated interaction of the enzyme with blue-Sepharose, homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli Top10 cells
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expression in Rhodococcus jostii RHA#2
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gene mhbM, DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
gene narX, DNA and amino acid sequence determination and analysis of the gentisate catabolic gene cluster. Among the 13 complete open reading frames deduced from this fragment, three narIKL encode the enzymes involved in the reactions of gentisate catabolism, of these, NarX is hydroxybenzoate 6-monooxygenase which converts 3-hydroxybenzoate to 2,5-dihydroxybenzoate. The narX gene is divergently transcribed with narIKL. Sequence comparisons, overview. Expression in of the His-tagged NarX in Escherichia coli strain Rosetta (DE3) pLysS
gene ncgl2923, sequence determination, analysis, and comparisons, functional overexpression in of N-terminally His-tagged Ncgl2923 in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
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)
orf2 or gene nagX, which does not encode salicylate 5-hydroxylase, but 3-hydroxybenzoate 6-hydroxylase, DNA and amino acid sequence determination and analysis, genetic organization, overview, overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R169E
site-directed mutagenesis, inactive mutant
H213D
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the mutant enzyme can bind to 3-hydroxybenzoate with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. It produces 2,5-dihydroxybenzoate with yields of 52%
H213E
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the mutant enzyme can bind to 3-hydroxybenzoate with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. It produces 2,5-dihydroxybenzoate with yields of 92%. The hydroxylation rate constant of the mutant enzyme (35/s) is similar to that of wild-type enzyme (36/s) and this variant has an efficiency of hydroxylation (92%) similar to the wild-type enzyme (86%)
Q301E
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inactive
Y105F
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the mutant shows less affinity for the aromatic substrate and lower catalytic efficiency compared to the wild type enzyme
Y217A
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the mutant enzyme does not show any perturbation of flavin absorption upon addition of 3-hydroxybenzoate
Y217F
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the mutant enzyme has a Kd value for 3-hydroxybenzoate binding of 7.5 mM, which is about 50fold larger than that found for wild-type enzyme. The results indicate that Tyr217 is necessary for substrate binding
Y217S
-
the mutant enzyme does not show any perturbation of flavin absorption upon addition of 3-hydroxybenzoate
additional information
Show AA Sequence (445 entries)
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