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Literature summary extracted from

  • Nianios, D.; Thierbach, S.; Steimer, L.; Lulchev, P.; Klostermeier, D.; Fetzner, S.
    Nickel quercetinase, a promiscuous metalloenzyme metal incorporation and metal ligand substitution studies (2015), BMC Biochem., 16, 10 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.24 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

EC Number Protein Variants Comment Organism
1.13.11.24 E76D site-directed mutagenesis, the mutant retains marginal activity Streptomyces sp. FLA
1.13.11.24 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
1.13.11.24 H69A site-directed mutagenesis, the mutant retains marginal activity Streptomyces sp. FLA
1.13.11.24 H71A site-directed mutagenesis, the mutant retains marginal activity Streptomyces sp. FLA
1.13.11.24 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

EC Number Localization Comment Organism GeneOntology No. Textmining
1.13.11.24 cytoplasm
-
 Streptomyces sp. FLA 5737
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.13.11.24 Co2+ activates, enzyme-bound Streptomyces sp. FLA
1.13.11.24 Fe2+ enzyme-bound, only poorly supports catalytic activity Streptomyces sp. FLA
1.13.11.24 Fe3+ enzyme-bound Streptomyces sp. FLA
1.13.11.24 Mn2+ activates, enzyme-bound Streptomyces sp. FLA
1.13.11.24 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
1.13.11.24 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
1.13.11.24 Zn2+ enzyme-bound Streptomyces sp. FLA

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.13.11.24 quercetin + O2 Streptomyces sp. FLA
-
 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.24 Streptomyces sp. FLA A2VA43
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.24 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

EC Number Source Tissue Comment Organism Textmining
1.13.11.24 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 [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.13.11.24 8
-
 purified recombinant Fe2+-supplemented enzyme, pH 8.0, 30°C Streptomyces sp. FLA
1.13.11.24 18
-
 purified recombinant Mn2+-supplemented enzyme, pH 8.0, 30°C Streptomyces sp. FLA
1.13.11.24 30
-
 purified recombinant Co2+-supplemented enzyme, pH 8.0, 30°C Streptomyces sp. FLA
1.13.11.24 137
-
 purified recombinant Ni2+-supplemented enzyme, pH 8.0, 30°C Streptomyces sp. FLA

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.24 quercetin + O2
-
 Streptomyces sp. FLA 2-(3,4-dihydroxybenzoyloxy)-4,6-dihydroxybenzoate + CO + H+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.13.11.24 dimer the enzyme is a dimer of monocupin subunits Streptomyces sp. FLA

Synonyms

EC Number Synonyms Comment Organism
1.13.11.24 Co-QueD
-
 Streptomyces sp. FLA
1.13.11.24 Fe-QueD
-
 Streptomyces sp. FLA
1.13.11.24 flavonol 2,4-dioxygenase
-
 Streptomyces sp. FLA
1.13.11.24 Mn-QueD
-
 Streptomyces sp. FLA
1.13.11.24 Ni-QueD
-
 Streptomyces sp. FLA
1.13.11.24 nickel quercetinase
-
 Streptomyces sp. FLA
1.13.11.24 QueD
-
 Streptomyces sp. FLA
1.13.11.24 quercetinase
-
 Streptomyces sp. FLA

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.13.11.24 30
-
 assay at Streptomyces sp. FLA

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.24 8
-
 assay at Streptomyces sp. FLA

General Information

EC Number General Information Comment Organism
1.13.11.24 evolution quercetinases are metal-dependent dioxygenases of the cupin superfamily Streptomyces sp. FLA
1.13.11.24 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
1.13.11.24 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