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Literature summary for 1.13.11.24 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)

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

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

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

Metals/Ions

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/ Products (Substrates)

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

Organism UniProt Comment Textmining
Streptomyces sp. FLA A2VA43
-
-

Purification (Commentary)

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

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 [micromol/min/mg]

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 and Products (Substrate)

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

Subunits

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

Synonyms

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

pH Optimum

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

General Information

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