Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | 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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0532 | - |
homogentisate | pH not specified in the publication, 25°C, wild-type enzyme | Pseudomonas putida |
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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | Q88E47 | - |
- |
Pseudomonas putida KT 2240 | Q88E47 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and hydroxyapatite chromatography | Pseudomonas putida |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
homogentisate + O2 = 4-maleylacetoacetate | reaction mechanism | Pseudomonas putida |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
homogentisate + O2 | - |
Pseudomonas putida | 4-maleylacetoacetate | - |
? | |
homogentisate + O2 | - |
Pseudomonas putida KT 2240 | 4-maleylacetoacetate | - |
? |
Synonyms | Comment | Organism |
---|---|---|
HGDO | - |
Pseudomonas putida |
homogentisate dioxygenase | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Pseudomonas putida |
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 | 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 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 |