Literature summary for 1.13.11.5 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.

Search for more literature for 1.13.11.5

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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)

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

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	Q88E47	-	-
Pseudomonas putida KT 2240	Q88E47	-	-

Purification (Commentary)

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

Reaction

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

Substrates and Products (Substrate)

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

Synonyms

Synonyms	Comment	Organism
HGDO	-	Pseudomonas putida
homogentisate dioxygenase	-	Pseudomonas putida

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

General Information

General Information	Comment	Organism
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
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
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]

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