Cloned (Comment) | Organism |
---|---|
gene queD, recombinant expression of C-terminally Strep-tagged wild-type and mutant enzymes in Escherichia coli in CoCl2- or MnCl2-supplemented medium | Streptomyces sp. FLA |
Protein Variants | Comment | Organism |
---|---|---|
E76D | site-directed mutagenesis, the mutant retains marginal activity | Streptomyces sp. FLA |
E76H | site-directed mutagenesis, the mutation results in Ni- and Co-QueD variants that retain the native fold and show residual catalytic activity | Streptomyces sp. FLA |
H69A | site-directed mutagenesis, the mutant retains marginal activity | Streptomyces sp. FLA |
H71A | site-directed mutagenesis, the mutant retains marginal activity | Streptomyces sp. FLA |
additional information | the metal-ligating amino acids for the structural integrity and function of Ni-QueD, the individual residues of the 3His/1Glu motif are replaced by site-directed mutagenesis | Streptomyces sp. FLA |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytoplasm | - |
Streptomyces sp. FLA | 5737 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Co2+ | activates, enzyme-bound | Streptomyces sp. FLA | |
Fe2+ | enzyme-bound, only poorly supports catalytic activity | Streptomyces sp. FLA | |
Fe3+ | enzyme-bound | Streptomyces sp. FLA | |
Mn2+ | activates, enzyme-bound | Streptomyces sp. FLA | |
additional information | the enzyme is metal-dependent. Cu2+ and Zn2+ do not support catalytic activity. Heterologous formation of catalytically active, native QueD holoenzyme requires Ni2+, Co2+ or Mn2+, i.e. metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA, metal content analysis of wild-type and mutant enzymes, detailed overview | Streptomyces sp. FLA | |
Ni2+ | activates best, enzyme-bound, a nickel quercetinase. Ni2+ ions support correct folding, the catalytic activity of wild-type QueD is likely mediated by a Ni2+ center | Streptomyces sp. FLA | |
Zn2+ | enzyme-bound | Streptomyces sp. FLA |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
quercetin + O2 | Streptomyces sp. FLA | - |
2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+ | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces sp. FLA | A2VA43 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally Strep-tagged wild-type and mutant enzymes from Escherichia coli to electrophoretic homogeneity by hydrophobic interaction chromatography, dialysis, two different steps of anion exchange chromatography, dialysis and ultrafiltration | Streptomyces sp. FLA |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
additional information | Streptomyces sp. strain FLA grows very poorly on quercetin as sole carbon source. The cells grow on medium supplemented with Mn2+, Zn2+, or Co2+ | Streptomyces sp. FLA | - |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
8 | - |
purified recombinant Fe2+-supplemented enzyme, pH 8.0, 30°C | Streptomyces sp. FLA |
18 | - |
purified recombinant Mn2+-supplemented enzyme, pH 8.0, 30°C | Streptomyces sp. FLA |
30 | - |
purified recombinant Co2+-supplemented enzyme, pH 8.0, 30°C | Streptomyces sp. FLA |
137 | - |
purified recombinant Ni2+-supplemented enzyme, pH 8.0, 30°C | Streptomyces sp. FLA |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
quercetin + O2 | - |
Streptomyces sp. FLA | 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+ | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | the enzyme is a dimer of monocupin subunits | Streptomyces sp. FLA |
Synonyms | Comment | Organism |
---|---|---|
Co-QueD | - |
Streptomyces sp. FLA |
Fe-QueD | - |
Streptomyces sp. FLA |
flavonol 2,4-dioxygenase | - |
Streptomyces sp. FLA |
Mn-QueD | - |
Streptomyces sp. FLA |
Ni-QueD | - |
Streptomyces sp. FLA |
nickel quercetinase | - |
Streptomyces sp. FLA |
QueD | - |
Streptomyces sp. FLA |
quercetinase | - |
Streptomyces sp. FLA |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Streptomyces sp. FLA |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Streptomyces sp. FLA |
General Information | Comment | Organism |
---|---|---|
evolution | quercetinases are metal-dependent dioxygenases of the cupin superfamily | Streptomyces sp. FLA |
malfunction | replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduces or abolishes quercetinase activity and affects its structural integrity. Only substitution of the glutamate ligand (E76) by histidine results in Ni- and Co-QueD variants that retain the native fold and show residual catalytic activity | Streptomyces sp. FLA |
physiological function | conversion of quercetin to 2-protocatechuoylphloroglucinol carboxylic acid is catalyzed by quercetinase, i.e. flavonol 2,4-dioxygenase. The the catalytic activity of wild-type QueD is likely mediated by a Ni2+ center | Streptomyces sp. FLA |