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Information on EC 1.13.11.5 - homogentisate 1,2-dioxygenase and Organism(s) Pseudomonas putida and UniProt Accession Q88E47

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IUBMB Comments
Requires Fe2+.
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This record set is specific for:
Pseudomonas putida
UNIPROT: Q88E47
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The taxonomic range for the selected organisms is: Pseudomonas putida
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
homogentisate 1,2-dioxygenase, homogentisic acid oxidase, homogentisate dioxygenase, homogentisate 1,2 dioxygenase, elhdo, homogentisate oxidase, homogentisic acid 1,2-dioxygenase, homogentisate oxygenase, homogentisate phytyl-transferase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homogentisate dioxygenase
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HGO
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-
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homogentisate dioxygenase
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-
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homogentisate oxidase
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-
-
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homogentisate oxygenase
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-
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homogentisic acid oxidase
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-
-
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homogentisic acid oxygenase
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-
-
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homogentisic oxygenase
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-
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homogentisicase
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-
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HTO
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-
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
homogentisate + O2 = 4-maleylacetoacetate
show the reaction diagram
reaction mechanism
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
redox reaction
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oxidation
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reduction
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SYSTEMATIC NAME
IUBMB Comments
homogentisate:oxygen 1,2-oxidoreductase (decyclizing)
Requires Fe2+.
CAS REGISTRY NUMBER
COMMENTARY hide
9029-49-6
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SUBSTRATE
PRODUCT                       
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
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-
-
?
homogentisate + O2
4-maleylacetoacetate
show the reaction diagram
-
-
-
-
ir
NATURAL SUBSTRATE
NATURAL PRODUCT
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
-
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
required, ferrous ion content of 0.56 mol of iron per mol of protein, all twelve subunits in the a.u. contain fully occupied Fe sites, octahedral coordination for Fe2+ with two histidine residues (His331 and His367), the enzyme uses a mononuclear nonheme Fe2+ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0532
homogentisate
pH not specified in the publication, 25°C, wild-type enzyme
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
79.5
homogentisate
pH not specified in the publication, 25°C, wild-type enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.6 - 5038
homogentisate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.74
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calculated value
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
despite different folds, active site architectures, and Fe2+ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediatesto catalyze the O2-dependent cleavage of aromatic rings. Convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments
physiological function
homogentisate 1,2-dioxygenase uses a mononuclear nonheme Fe2+ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate, i.e 2,5-dihydroxyphenylacetate
additional information
the active site pocket with its Fe2+ ion is freely accessible from the outside through a wide opening. Homogentisate binds as a monodentate ligand to Fe2+, and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates, i.e. superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates. central role of Y346 in substrate binding and turnover
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
48000
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SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kim, Y.H.; Cho, K.; Yun, S.H.; Kim, J.Y.; Kwon, K.H.; Yoo, J.S.; Kim, S.I.
Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis
Proteomics
6
1301-1318
2006
Pseudomonas putida, Pseudomonas putida KT 2440
Manually annotated by BRENDA team
Jeoung, J.H.; Bommer, M.; Lin, T.Y.; Dobbek, H.
Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase
Proc. Natl. Acad. Sci. USA
110
12625-12630
2013
Pseudomonas putida (Q88E47), Pseudomonas putida KT 2240 (Q88E47)
Manually annotated by BRENDA team