Information on EC 1.13.11.4 - gentisate 1,2-dioxygenase

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

EC NUMBER
COMMENTARY hide
1.13.11.4
-
RECOMMENDED NAME
GeneOntology No.
gentisate 1,2-dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2,5-dihydroxybenzoate + O2 = maleylpyruvate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
2,5-xylenol and 3,5-xylenol degradation
-
-
gentisate degradation I
-
-
gentisate degradation II
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
salicylate degradation IV
-
-
Tyrosine metabolism
-
-
3-phenylpropionate degradation
-
-
SYSTEMATIC NAME
IUBMB Comments
gentisate:oxygen 1,2-oxidoreductase (decyclizing)
Requires Fe2+.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-48-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain RES167
-
-
Manually annotated by BRENDA team
strain RES167
-
-
Manually annotated by BRENDA team
strain O157:H7
SwissProt
Manually annotated by BRENDA team
strain D1
-
-
Manually annotated by BRENDA team
strain D1
-
-
Manually annotated by BRENDA team
strain M5a1
-
-
Manually annotated by BRENDA team
strain NCIB 9867
-
-
Manually annotated by BRENDA team
strain NCIMB 9867
-
-
Manually annotated by BRENDA team
strain NCIB 9869
-
-
Manually annotated by BRENDA team
strains C4, C5, and C6
-
-
Manually annotated by BRENDA team
strain GMI 1000
-
-
Manually annotated by BRENDA team
strain GMI 1000
-
-
Manually annotated by BRENDA team
strain U2
-
-
Manually annotated by BRENDA team
strain U2
-
-
Manually annotated by BRENDA team
gene genH
UniProt
Manually annotated by BRENDA team
gene genH
UniProt
Manually annotated by BRENDA team
gene narI
UniProt
Manually annotated by BRENDA team
gene narI
UniProt
Manually annotated by BRENDA team
DSS-3
SwissProt
Manually annotated by BRENDA team
RW5
-
-
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
1,4-dihydroxy-2-naphthoate + O2
?
show the reaction diagram
1-hydroxy-2-naphthoate + O2
?
show the reaction diagram
2,5-dihydroxybenzoate + O2
maleylpyruvate
show the reaction diagram
2,5-xylenol + O2
?
show the reaction diagram
3,4-dichlorosalicylate + O2
?
show the reaction diagram
3,4-dimethylgentisate + O2
?
show the reaction diagram
3,5-dibromosalicylate + O2
?
show the reaction diagram
3,6-dichlorogentisate + O2
?
show the reaction diagram
3-aminosalicylate + O2
?
show the reaction diagram
3-bromogentisate + O2
3-bromo-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-bromosalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
3-chlorogentisate + O2
3-chloro-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
-
-
-
-
?
3-chlorosalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
3-ethylgentisate + O2
3-ethyl-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-fluorogentisate + O2
3-fluoro-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-hydroxybenzoate + O2
3,4-dihydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-hydroxybenzoate + O2
?
show the reaction diagram
3-hydroxysalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
3-isopropyl-gentisate + O2
4-hydroxy-3-isopropyl-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-isopropylgentisate + O2
4-hydroxy-3-isopropyl-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-methylgentisate + O2
3-methyl-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
-
Vmax is 10.1% of Vmax for gentisate
-
-
?
3-methylgentisate + O2
4-hydroxy-3-methyl-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
3-methylsalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
3-O-methylgentisate + O2
?
show the reaction diagram
4-bromosalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
4-chlorogentisate + O2
2-chloro-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
4-chlorosalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
4-fluorogentisate + O2
2-fluoro-4-hydroxy-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
4-hydroxybenzoate + O2
?
show the reaction diagram
4-hydroxysalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
4-isopropylgentisate + O2
4-hydroxy-2-isopropyl-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
4-methylgentisate + O2
4-hydroxy-2-isopropyl-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
-
-
-
-
?
4-methylgentisate + O2
4-hydroxy-2-methyl-6-oxo-hepta-2,4-dienedioic acid
show the reaction diagram
4-methylsalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
4-O-methylgentisate + O2
?
show the reaction diagram
5-aminosalicylate + O2
?
show the reaction diagram
5-bromosalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
5-chlorosalicylate + O2
?
show the reaction diagram
-
kcat/KM is less than 1% of the kcat/Km for gentisate
-
-
?
5-fluorosalicylate + O2
?
show the reaction diagram
5-methylsalicylate + O2
?
show the reaction diagram
benzoate + O2
3-hydroxy-6-oxoheptadienoic acid
show the reaction diagram
-
-
-
-
?
cinnamate + O2
?
show the reaction diagram
-
-
-
-
?
gentisate + O2
?
show the reaction diagram
gentisate + O2
maleylpyruvate
show the reaction diagram
lactate + O2
?
show the reaction diagram
phenylpropionate + O2
?
show the reaction diagram
-
-
-
-
?
salicylate + O2
2-oxohepta-3,5-dienedioic acid
show the reaction diagram
salicylate + O2
?
show the reaction diagram
-
-
-
-
?
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
2,5-dihydroxybenzoate + O2
maleylpyruvate
show the reaction diagram
gentisate + O2
maleylpyruvate
show the reaction diagram
salicylate + O2
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
optimal salt concentration is 2 M KCl or NaCl
NaCl
optimal salt concentration is 2 M KCl or NaCl
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-hydroxy 2 naphthoate
0.33 mM, 19% of wild type enzyme activity remaining
2,2'-dipyridyl
3-hydroxybenzoate
-
-
Ca2+
-
5 mM, partial inhibition
Fe
2 mM, complete inactivation
Fe2+
-
1 mM, complete inactivation
H2O2
-
3 mM, 90% inactivation
iodoacetic acid
-
-
Li2SO4
5 mM 5-10% inhibition at pH 8.0
Mg2+
-
66% residual activity at 5 mM
MgSO4
5 mM, 5-10% inhibition at pH 8.0
Na+
-
5 mM, partial inhibition
Na2SO4
5 mM, 5-10% inhibition at pH 8.0
o-phenanthroline
salicylate
Sodium arsenate
-
-
Thiosalicylate
-
-
Tiron
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
ascorbate
dithiothreitol
glycerol
L-cysteine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.045 - 0.29
1,4-dihydroxy-2-naphthoate
0.0209
1-hydroxy 2 naphthoate
-
0.134
1-hydroxy-2-naphthoate
-
-
0.14
1-hydroxynaphthalene-2-carboxylic acid
-
wild-type enzyme, pH and temperature not specified in the publication
0.011 - 0.113
2,5-Dihydroxybenzoate
0.285
3,4-dichlorosalicylate
-
pH 8.0
0.14 - 7.7
3,4-Dimethylgentisate
0.094
3,5-Dibromosalicylate
-
pH 8.0
0.754
3,6-dichlorogentisate
-
-
2.3
3-aminosalicylate
-
pH 8.0
0.011 - 0.33
3-Bromogentisate
0.071
3-bromosalicylate
-
pH 8.0
0.027
3-chlorosalicylate
-
pH 8.0
0.01155 - 3.65
3-Ethylgentisate
0.035 - 0.08962
3-Fluorogentisate
0.067
3-hydroxysalicylate
-
pH 8.0
0.0043 - 2.75
3-Isopropylgentisate
0.027 - 1.75
3-Methylgentisate
0.116
3-methylsalicylate
-
pH 8.0
1.18
3-O-Methylgentisate
-
-
0.02326 - 0.24
4-Chlorogentisate
0.1
4-chlorosalicylate
-
pH 8.0
0.015 - 0.044
4-Fluorogentisate
0.039
4-hydroxysalicylate
-
pH 8.0
1.2 - 1.23
4-Isopropylgentisate
0.057 - 0.52
4-Methylgentisate
0.028
4-methylsalicylate
-
pH 8.0
0.45
4-O-Methylgentisate
-
-
0.216 - 3.1
5-Aminosalicylate
0.126
5-bromosalicylate
-
pH 8.0
0.085
5-chlorosalicylate
-
pH 8.0
0.047 - 0.091
5-Fluorosalicylate
0.328 - 0.788
5-Methylsalicylate
0.0071 - 0.167
gentisate
0.055 - 0.096
O2
0.012 - 0.017
salicylate
additional information
additional information
-
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.565 - 6.08
1-hydroxy 2 naphthoate
44
1-hydroxy-2-naphthoate
Pseudaminobacter salicylatoxidans
-
-
24
1-hydroxynaphthalene-2-carboxylic acid
Pseudaminobacter salicylatoxidans
-
wild-type enzyme, pH and temperature not specified in the publication
5.5
2,5-Dihydroxybenzoate
Xanthobacter polyaromaticivorans
Q75W71
-
0.2
3,4-dichlorosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.02
3,5-Dibromosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
4
3-aminosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.3
3-bromosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.4
3-chlorosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.5
3-hydroxysalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
2.9
3-methylsalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.7
4-bromosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
-
0.8
4-chlorosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.2
4-hydroxysalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.3
4-methylsalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
2.8 - 59.3
5-Aminosalicylate
0.1
5-bromosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
0.5
5-chlorosalicylate
Pseudaminobacter salicylatoxidans
-
pH 8.0
2.4 - 3.6
5-Fluorosalicylate
2.1 - 14.3
5-Methylsalicylate
94 - 642
gentisate
0.9 - 2.3
salicylate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.04
-
23ºC, with gentistate as substrate, G56 mutant
0.08
-
23ºC, with lactate as substrate, wild-type enzyme
0.09
-
23ºC, with 2,5-xylenol as substrate, G56 mutant
0.11
-
23ºC, with 3-hydroxybenzoate as substrate, G56 mutant
0.45
-
crude extract, at 23°C
0.52
-
23ºC, with gentistate as substrate, wild-type enzyme
0.72
-
crude extract, at 23°C
0.75
-
23ºC, with 2,5-xylenol as substrate, wild-type enzyme
0.81
-
23ºC, with 3-hydroxybenzoate as substrate, wild-type enzyme
1.2
pH 7.0, 22°C, extract from cells grown with benzoic acid
1.4
pH 7.0, 22°C, extract from cells grown with 3-phenylpropionic acid
1.5
pH 7.0, 22°C, extract from cells grown with cinnamic acid
23.2
-
after 51.56fold purification, at 23°C
43.14
-
after 59.92fold purification, at 23°C
145
recombinant enzyme, pH 7.4, 30°C
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9.4
-
activity range, isozymes Nagl2 and Nagl3
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15
-
no activity below
25 - 50
-
isozyme Nagl2, 74.1% activity at 25°C, maximum activity at 50°C
25 - 65
-
isozyme Nagl4, 72.4% activity at 25°C, maximum activity at 65°C
60
-
no activity above
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 4.7
-
pH range 3.5-9.5
4.6 - 4.8
4.85
-
isoelectric focusing
6.8
-
isozyme Nagl1, sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36500
4 * 36500, gel filtration
38900
SDS-PAGE
39000
-
4 * 39000, SDS-PAGE
39300
calculated from amino acid sequence
40800
-
4 * 40800, SDS-PAGE
41000
-
2 * 41000; 2 * 41000, gel filtration
41176
-
x * 41176, calculated from sequence
42000
-
4 * 42000, SDS-PAGE
43000
-
8 * 43000, SDS-PAGE
44000
-
4 * 44000, gel filtration
82000
-
gel filtration
132000
-
gel filtration
150000
-
non-denaturing PAGE
154000
158000
-
gel filtration
159000
-
gel filtration
164000
-
gel filtration
174000
-
gel filtration
177000
-
gel filtration, recombinant protein from Escherichia coli
185000
-
gel filtration
328000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homodimer
homotetramer
octamer
-
8 * 43000, SDS-PAGE
tetramer
trimer
-
3 * 45000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapour diffusion method using 0.1 M BisTris pH 6.5 and 20% polyethylene glycol 5000 monomethylether
diffraction-quality crystals of salicylate 1,2-dioxygenase are obtained using the sitting-drop vapour-diffusion method at 4°C from a solution containing 10%(w/v) ethanol, 6%(w/v) PEG 400, 0.1 M sodium acetate pH 4.6. Crystals belong to the primitive tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = 133.3, c = 191.51 A
-
purified G106A enzyme mutant free and complexes with gentisate and salicylate, crystallization under anaerobic conditions, sitting drop vapor diffusion method, mixing of 0.001 ml of protein solution with 0.0008 ml of 8% poly(ethylene glycol)10000 and 0.1 M Tris/HCl pH 8.0, and 0.0002 ml of 0.1 M calcium chloride, crystal quality is improved using the seeding technique, X-ray diffraction structure determination and analysis at 2.0-2.58 A resolution
-
crystal structure at 2.8 A resolution, hanging drop vapor diffusion crystallization at 16°C. The crystal form belongs to the R32 space group, with two protein molecules (named as A and B) per asymmetric unit and 45% solvent content
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 7.5
-
-
389996
6.5
GDO loses its activity after being kept in buffer below pH 6.5 for 12 h in the absence of ferrous iron and cysteine
672692
7.2
-
-
389982
8
GDO loses its activity after being kept in buffer above pH 8.0 for 12 h in the absence of ferrous iron and cysteine
672692
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
-
80 min, 50% loss of activity
70
-
protein unfolds and loses its secondary structures above
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
cysteine stabilizes
Fe2+ stabilizes
glycerol stabilizes
-
glycerol, beta-mercaptoethanol stabilize
-
highly unstable in 50 mM MOPS buffer with no additives
purified GDO proteins NagI2 and NagI3 are highly unstable in phosphate buffer without ferrous ions
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
exposure of enzyme to oxidants, e.g. H2O2 or K3Fe(CN)6, at 1 mM concentrations results in complete loss of activity, even under anaerobic conditions
under turnover conditions enzyme is rapidly inactivated at O2 concentrations above 0.8 mM
-
389987
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 1 week, purified enzyme loses 51% of its initial activity
-
-20°C, addition of glycerol and beta-mercaptoethanol, 96 h, 25% of activity remains
-
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 12 months, 3.5% residual activity
-
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 12 months, 42.9% residual activity
-
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 3 months, 42.5% residual activity
-
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 3 months, 86.5% residual activity
-
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 6 months, 23.1% residual activity
-
-20°C, MOPS buffer with 0.1 mM Fe2+, 2 mM L-cysteine, and 10% (v/v) glycerol, 6 months, 70.9% residual activity
-
-70°C, 1 week, purified enzyme loses 30% of its initial activity
-
-70°C, 2 weeks, 70% loss of activity
-
-80°C, storage following rapid freezing in liquid N2, protein concentration above 10 mg/ml, about 15% loss of activity after 1 year
4°C, 48 h, complete loss of activity
-
4°C, 72 h, 90% loss of activity
-
4°C, MOPS buffer with no additives, 24 h, 22.5% residual activity
-
4°C, MOPS buffer with no additives, 24 h, 33.9% residual activity
-
4°C, MOPS buffer with no additives, 96 h, complete loss of activity
cell free extracts adsorbed on polyester filters, stable
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; ammonium sulfate fractionation, DEAE-Sepharose fast flow column chromatography
GSTrap fast flow column chromatography
-
His-Bind protein chromatography
-
HiTrapQ column chromatography
nickel-nitriloacetic acid resin column chromatography
recombinant enzyme
recombinant mutant G106A
-
recombinant Nagl2 and Nagl3 isozymes from Escherichia coli strain BL21(DE3) by gel filtration and sequential anion exchange chromatography
-
recombinant protein from Escherichia coli
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
deduced amino acid sequence contains 4 novel histidine clusters and a possible extradiol fingerprint region
-
expressed in Escherichia coli
expressed in Escherichia coli BL21 cells
-
expressed in Escherichia coli Rosetta cells
expressed in Escherichia coli strain BL21/pET28ancg12920
-
expression in Escherichia coli
expression of wild-type and mutant enzymes
-
gene dbdB, DNA and amino acid sequence determination and analysis, genetic organization, phylogenetic analysis, overview
gene genH, DNA and amino acid sequence determination and analysis, gene genH belongs to the gen gene cluster, RT-PCR expression and genomic analysis, overview
gene narI, 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, NarI is gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate. Genes narI, narK, and narL genes are transcribed as a single operon only in the presence of naphthalene. Sequence comparisons, overview. Expression in Escherichia coli strain Rosetta (DE3) pLysS
genes nagI1, nagI2, and nagI3, the latter encoded in the third cluster, nagl3 is polycistronic and is expressed at a relatively high level, genetic organization, DNA and amino acid sequence determination and analysis, real-time RT-PCR for gene expression analysis of genes nagI1, nagI2 and nagI3 and the third catabolic gene cluster, overview. Overexpression of all three genes in Escherichia coli strain BL21(DE3). The transformation efficiency of the pET21bnagI1 ligation product is very low compared with that of the nagI2 and nagI3 genes
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overexpressed as a hexahistidine fusion protein from Escherichia coli
-
overexpressed in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
nagl3 gene expression is induced by salicylate in wild-type strain CJ2 but not in the nagR knockout mutant strain CJN110
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
H108D
-
complete loss of activity
H110D
-
complete loss of activity
H118D
-
complete loss of activity
H120D
-
complete loss of activity
H149D
-
complete loss of activity
H151D
-
complete loss of activity
H159D
-
complete loss of activity
H161D
-
complete loss of activity
H108D
-
complete loss of activity
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H110D
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complete loss of activity
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H149D
-
complete loss of activity
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H151D
-
complete loss of activity
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H161D
-
complete loss of activity
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F159Y
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site-directed mutagenesis
G106A
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site-directed mutagenesis, in contrast to the wild-type enzyme, mutant G106A oxidizes only gentisate, while 1-hydroxy-2-naphthoate and salicylate are not converted. The mutant shows slightly decreased activity with salicylate, but shows a higher affinity to this substratecompared to the wild-type. Salicylate is coordinated in the G106A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that is formed between Trp104 and Asp174 in the G106A variant
G111A
-
site-directed mutagenesis
M103L
-
site-directed mutagenesis
R113G
-
site-directed mutagenesis
S147R
-
site-directed mutagenesis
G106A
-
site-directed mutagenesis, in contrast to the wild-type enzyme, mutant G106A oxidizes only gentisate, while 1-hydroxy-2-naphthoate and salicylate are not converted. The mutant shows slightly decreased activity with salicylate, but shows a higher affinity to this substratecompared to the wild-type. Salicylate is coordinated in the G106A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that is formed between Trp104 and Asp174 in the G106A variant
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G111A
-
site-directed mutagenesis
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M103L
-
site-directed mutagenesis
-
R113G
-
site-directed mutagenesis
-
S147R
-
site-directed mutagenesis
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D120K
-
complete loss of activity
E223A
-
102% relative activity compared to the wild type enzyme
G123N
-
75% relative activity compared to the wild type enzyme
G164T
-
complete loss of activity
H108D
-
no enzymatic activity
H110D
-
no enzymatic activity
H149D
-
no enzymatic activity
H151D
-
no enzymatic activity
K338Y
-
156% relative activity compared to the wild type enzyme
M146T
-
complete loss of activity
M169T
-
complete loss of activity
N153H
-
183% relative activity compared to the wild type enzyme
N43T
-
317% relative activity compared to the wild type enzyme
S113P
-
complete loss of activity
T260C
-
complete loss of activity
V326Q
-
complete loss of activity
V36A
-
complete loss of activity
Y17C
-
161% relative activity compared to the wild type enzyme
Y181D
-
68% relative activity compared to the wild type enzyme
Y181F
-
mutant demonstrates 4-, 3-, 6-, and 16fold increase in relative activity towards 2,5-dihydroxybenzoate, 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively, and shows 464% relative activity compared to the wild type enzyme for 2,5-dihydroxybenzoate
Y181H
-
98% relative activity compared to the wild type enzyme
Y284I
-
260% relative activity compared to the wild type enzyme
D120K
-
complete loss of activity
-
G123N
-
75% relative activity compared to the wild type enzyme
-
N43T
-
317% relative activity compared to the wild type enzyme
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Y17C
-
161% relative activity compared to the wild type enzyme
-
Y181F
-
mutant demonstrates 4-, 3-, 6-, and 16fold increase in relative activity towards 2,5-dihydroxybenzoate, 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively, and shows 464% relative activity compared to the wild type enzyme for 2,5-dihydroxybenzoate
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H108D
-
complete loss of activity
H110D
-
complete loss of activity
H118D
-
complete loss of activity
H120D
-
complete loss of activity
H149D
-
complete loss of activity
H151D
-
complete loss of activity
H159D
-
complete loss of activity
H161D
-
complete loss of activity
H108D
-
complete loss of activity
-
H110D
-
complete loss of activity
-
H149D
-
complete loss of activity
-
H151D
-
complete loss of activity
-
H161D
-
complete loss of activity
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D175N
-
inactive mutant enzyme
D225N
-
activity is comparable to wild-type activity
E47Q
-
activity is comparable to wild-type activity
H119A/H121A
-
despite its wild-type like structural propertie, the mutant shows extremely low gentisate 1,2-dioxygenase activity
H290A/H292A
-
mutant can not be expressed in a soluble form
L39T
-
activity is comparable to wild-type activity
P253_Y254del
-
mutation reduces the activity to 30%
Q108E
-
inactive mutant enzyme
Q108E/D175N
-
activity is near zero
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell free extracts adsorbed on polyester filters, reactivation using ferrous iron and ascorbate
-
H2O2-inactivated enzyme is restored by hydroxylamine or ferrous iron
-
Show AA Sequence (236 entries)
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