Information on EC 1.13.11.5 - homogentisate 1,2-dioxygenase

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

EC NUMBER
COMMENTARY hide
1.13.11.5
-
RECOMMENDED NAME
GeneOntology No.
homogentisate 1,2-dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
homogentisate + O2 = 4-maleylacetoacetate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
L-tyrosine degradation I
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
Styrene degradation
-
-
tyrosine metabolism
-
-
Tyrosine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
homogentisate:oxygen 1,2-oxidoreductase (decyclizing)
Requires Fe2+.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-49-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Agave toumeyana
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain biA1
Uniprot
Manually annotated by BRENDA team
UBC 814
-
-
Manually annotated by BRENDA team
UBC 814
-
-
Manually annotated by BRENDA team
Bos taurus Chinese red cattle
-
UniProt
Manually annotated by BRENDA team
Burkholderia xenovorans
genes hmgA1 and hmgA2
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
var. nanus
-
-
Manually annotated by BRENDA team
Pigeon
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
synthesis of homogentisate-1,2-dioxygenase (HmgA) is highly induced in anaerobic norCB bacteria, and not detected in wild-type extracts
-
-
Manually annotated by BRENDA team
strain O6
-
-
Manually annotated by BRENDA team
strain O6
-
-
Manually annotated by BRENDA team
strain KT 2440
-
-
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Vitis vinifera x Vitis riparia
-
-
-
Manually annotated by BRENDA team
Vitis vinifera x Vitis vinifera
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
-
the enzyme is involved in tyrosine catabolism
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
3-chlorohomogentisate + O2
3-chloro-4-maleylacetoacetate
show the reaction diagram
-
1% of activity with homogentisate
-
-
?
3-methylhomogentisate + O2
3-methyl-4-maleylacetoacetate
show the reaction diagram
-
10% of activity with homogentisate
-
-
?
homogentisate + O2
4-fumarylacetoacetate
show the reaction diagram
homogentisate + O2
4-maleylacetoacetate
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
homogentisate + O2
4-maleylacetoacetate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
-
inactivation in the absence of O2 due to formation of a white precipitate corresponding to apoprotein
2,2'-dipyridyl
2-hydroxyphenylacetate
-
strong competitive inhibition with respect to homogentisate
2-mercaptoethanol
-
-
3,4-Dihydroxyphenylacetate
-
1 mM, 48% inhibition, substrate analogue
3-chlorohomogentisate
-
inactivation in the presence of O2 due to formation of a white precipitate corresponding to apoprotein
3-hydroxyphenylacetate
-
competitive inhibition with respect to homogentisate
ascorbate
-
strong inhibitory effect, but stabilizes
ascorbic acid
-
38% inhibition at pH 6.2
benzoquinoneacetic acid
-
-
-
CaCl2
-
0.001 mM, 56% inhibition
Co2+
-
incubation with Fe2+ plus Co2+ in equimolar concentrations inhibits
Cu2+
-
incubation with Fe2+ plus Cu2+ in equimolar concentrations inhibits
cyanide
cysteic acid
-
weak inhibition
cysteine
-
inhibition not reversed by glutathione
cystine
-
inhibition not reversed by glutathione
diphosphate
-
-
FeSO4
-
0.25 mM, 42% inhibition
glutathione
-
reduced glutathione slightly inhibits
Hg2+
-
inhibition reversed by glutathione
HgCl2
-
20 mM, complete inhibition
Lewisite
-
inhibits by competing with Fe2+ for a common enzymic binding site
Methylmercuric bromide
-
inhibits by competing with Fe2+ for a common enzymic binding site
NaCl
-
0.001 mM, 63% inhibition
Ni2+
-
incubation with Fe2+ plus Ni2+ in equimolar concentrations inhibits
p-chloromercuribenzoate
p-hydroxyphenylacetic acid
-
80 mM, complete inhibition, substrate analogue
p-hydroxyphenylpyruvate
-
1 mM, 68% inhibition, substrate analogue
phosphate
-
-
sulfhydryl reagents
-
strong inhibition
Superoxide dismutase
-
inhibits the reaction indicating the involvement of superoxide anions in the dioxygenase reaction
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Zn2+
-
incubation with Fe2+ plus Zn2+ in equimolar concentrations inhibits
additional information
-
not inhibited by Ca2+ and Mn2+
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-Hydroxy-3-methoxybenzaldehyde
-
2 mM
ascorbate
cysteine
glutathione
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.009 - 0.6
homogentisate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.53
3-chlorohomogentisate
Homo sapiens
-
25C, pH 6.2
5.3
3-methylhomogentisate
Homo sapiens
-
25C, pH 6.2
10.1 - 79.5
homogentisate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6 - 5038
homogentisate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.495
-
-
0.625
Agave toumeyana
-
-
0.75 - 1
-
value in crude lysates
28.3
-
recombinant HGO
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.2
-
Good buffer
7
Burkholderia xenovorans
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
approx. 15% of maximal activity at pH 5.0 and pH 8.0, respectively
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.74
-
calculated value
6.7
-
theoretical value
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
expression of HGO gene
Manually annotated by BRENDA team
Burkholderia xenovorans
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-
Manually annotated by BRENDA team
-
liver from a 20 week old male embryo
Manually annotated by BRENDA team
-
expression of HGO gene
Manually annotated by BRENDA team
-
expression of HGO gene
Manually annotated by BRENDA team
-
expression of HGO gene
Manually annotated by BRENDA team
-
expression of HGO gene
Manually annotated by BRENDA team
-
expression of HGO gene
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
-
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pseudomonas putida (strain KT2440)
Pseudomonas putida (strain KT2440)
Pseudomonas putida (strain KT2440)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
48000
-
SDS-PAGE
52900
-
theoretical value
149000
-
gel filtration
150000
-
-
199000
gel filtration
200000
-
gel filtration
254000
-
-
380000
-
ultracentrifugal analysis
424000 - 478000
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equilibrium sedimentation studies
450000 - 480000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexamer
homohexamer
-
or dimer of trimer, X-ray crystallography
monomer
tetramer
4 * 49750, gel filtration
trimer
-
3 * 49000, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
-
enzyme is not glycosylated
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures of apo-HGO and HGO containing an iron
-
three crystal structures of the enzyme, revealing five different steps in its reaction cycle, crystallization of purifed recombinant wild-type aand mutant enzymes, mixing of 15 mg/ml protein solution with an equal volume of reservoir solution containing 16-17% PEG 3350, 0.02 M Na,K phosphate, and 0.1 M Bis-Tris propane, pH 8.0, with or without 2 mM homogentisate, vapour diffusion method, X-ray diffraction structure determination and analysis at 1.7-1.98 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 7.2
-
-20C, stable for several weeks
439380
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 55
-
almost linear decrease of activity
37
-
50% loss of activity after 17 s, pH 7.0; 50% loss of activity after 28 s, pH 6.2
45
-
65% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
10% acetone stabilizes
-
ascorbate stabilizes, it probably protects Fe2+ from spontaneous oxidation
-
rather stable enzyme, can be repeatedly thawed and frozen without significant loss of activity
-
unlike the mammalian enzyme, the bacterial enzyme is fairly stable on aging or during storage
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
Fe2+, organic mercurials and reducing agents protect the enzyme from irreversible aerobic oxidation
-
396333
inactivation by air probably causes degradation of enzyme in two proteins
-
439390
sensitive to O2, easily inactivated by aeration
-
439380
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 50 mM potassium phosphate buffer, pH 7.0, crude extracts, 5 months, 25% loss of activity
-
-20C, in absence of Fe2+, about 3 days, stable
-
-20C, pH 6.0-7.2, in absence of Fe2+, several weeks, stable
-
-80, 20 mM Hepps, pH 8.0, 100 mM NaCl, 4 months, no loss of activity
-
4C, 20 mM Tris-Cl pH 7.8, 0.5 M NaCl, 0.3 M imidazole, 24 h, loses two third of its activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
19.2fold purification
-
190fold purification
-
330fold purification
-
54.1fold purification
-
nickel chelate affinity chromatography
partial purification
purification of a fusion protein of homogentisate dioxygenase and glutathione S-transferase, expressed in Escherichia coli
recombinant His-tagged HGO, Ni-affinity, Mono Q
-
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography
Superdex gel filtration and Q-Sepharose column chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
complete HGO cDNA, encoding for a 50 kDa protein, is cloned and sequenced
-
DNA and amino acid sequence determination and analysis, genotyping, polymorphism of the HGD gene in the exon 1 and intron 1
DNA and ammino acid sequence determinaation and analysis, located on chromosome 3(q23-3q21), genotyping and identification of narurally occuring mutations involved in alkaptonuria, overview
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expressed in Escherichia coli
expression of His-tagged HGO in Escherichia coli
-
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
gene HGD, DNA and amino acid sequence determination and analysis, genotyping, semi-quantitative RT-PCR analysis. Identification of nine single nucleotide polymorphisms, five in the coding region and four intronic
genes hmgA1 and hmgA2, the gene cluster hmgABC is located at C1 in strain LB400 genome, quantitative expression analysis
Burkholderia xenovorans
-
genotyping
-
HGO gene on chromosome 3 is cloned, completely sequenced and characterized, identification of its promoter region, transcriptional control of the gene
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hmgA gene encoding homogentisate dioxygenase, a 448 residue polypeptide, is cloned, sequenced and characterized, gene is expressed in Escherichia coli as a fusion to glutathione S-transferase
PCR of coding region of DNA extracted from EDTA blood sample
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
induction of fumarylacetoacetate hydrolase HmgB in Burkholderia xenovorans LB400 during growth on hydroxyphenylacetates
Burkholderia xenovorans
-
LB400 cells grown with 3-hydroxyphenylacetate degrade homogentisate and show homogentisate 1,2-dioxygenase activity. The hmgA1 gene is upregulated by 3-hydroxyphenylacetate in strain LB400. The expression of the hmgA2 gene is induced in 3-hydroxyphenylacetategrown-cells
Burkholderia xenovorans
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C1273A
-
naturally occuring mutation, the mutation is involved in alkaptonuria
G161R
-
naturally occuring mutation in the HGD gene, resulting in a specific genotype appearing in a Hungarian population, originating from Slovakia, with alkaptonuria, phenotype overview
G360R
-
active site mutation in exon 13
K57N
-
active site mutation in exon 3
T1046G
-
naturally occuring mutation, the mutation is involved in alkaptonuria
T533G
-
naturally occuring mutation, the mutation is involved in alkaptonuria
T847C
-
naturally occuring mutation, the mutation is involved in alkaptonuria
H288Q
site-directed mutagenesis, the mutant shows a 75fold reduction in kcat compared to the wild-type enzyme
Y346F
site-directed mutagenesis, replacement of Y346 by phenylalanine decreases the affinity for homogentisate more than 60fold and reduces the apparent kcat 20fold resulting in a decrease of the specificity constant by three orders of magnitude compared to the wild-type enzyme
H288Q
-
site-directed mutagenesis, the mutant shows a 75fold reduction in kcat compared to the wild-type enzyme
-
Y346F
-
site-directed mutagenesis, replacement of Y346 by phenylalanine decreases the affinity for homogentisate more than 60fold and reduces the apparent kcat 20fold resulting in a decrease of the specificity constant by three orders of magnitude compared to the wild-type enzyme
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
-
crude enzyme preparations can be used for a spectrophotometric method for routine, sensitive determination of homogentisate in human urine
medicine
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