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Literature summary extracted from

  • 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 (2013), Proc. Natl. Acad. Sci. USA, 110, 12625-12630.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.5 expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Pseudomonas putida

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.13.11.5 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 Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.5 H288Q site-directed mutagenesis, the mutant shows a 75fold reduction in kcat compared to the wild-type enzyme Pseudomonas putida
1.13.11.5 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 Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.5 0.0532
-
 homogentisate pH not specified in the publication, 25°C, wild-type enzyme Pseudomonas putida

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.5 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 Pseudomonas putida

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.5 homogentisate + O2 Pseudomonas putida
-
 4-maleylacetoacetate
-
 ?
1.13.11.5 homogentisate + O2 Pseudomonas putida KT 2240
-
 4-maleylacetoacetate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.5 Pseudomonas putida Q88E47
-
-
1.13.11.5 Pseudomonas putida KT 2240 Q88E47
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.5 recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography Pseudomonas putida

Reaction

EC Number Reaction Comment Organism Reaction ID
1.13.11.5 homogentisate + O2 = 4-maleylacetoacetate reaction mechanism Pseudomonas putida
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.5 homogentisate + O2
-
 Pseudomonas putida 4-maleylacetoacetate
-
 ?
1.13.11.5 homogentisate + O2
-
 Pseudomonas putida KT 2240 4-maleylacetoacetate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.13.11.5 HGDO
-
 Pseudomonas putida
1.13.11.5 homogentisate dioxygenase
-
 Pseudomonas putida

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.5 25
-
 assay at Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.11.5 79.5
-
 homogentisate pH not specified in the publication, 25°C, wild-type enzyme Pseudomonas putida

General Information

EC Number General Information Comment Organism
1.13.11.5 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 Pseudomonas putida
1.13.11.5 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 Pseudomonas putida
1.13.11.5 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 Pseudomonas putida

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.13.11.5 4.6
-
 homogentisate pH not specified in the publication, 25°C, mutant Y346F Pseudomonas putida
1.13.11.5 5038
-
 homogentisate pH not specified in the publication, 25°C, wild-type enzyme Pseudomonas putida