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Literature summary extracted from

  • Valley, M.P.; Brown, C.K.; Burk, D.L.; Vetting, M.W.; Ohlendorf, D.H.; Lipscomb, J.D.
    Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis (2005), Biochemistry, 44, 11024-11039.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.13.11.3 the enzyme containing the Y408E mutation is expressed in Pseudomonas fluorescens Pseudomonas putida
1.13.11.3 the enzymes containing the Y408C, Y408F and Y408H mutation are expressed in Escherichia coli Pseudomonas putida

Protein Variants

EC Number Protein Variants Comment Organism
1.13.11.3 Y408C iron is tightly bound. The structure reveals no significant mutation-related changes except in the immediate vicinity of the altered amino acid (rmsd over all atoms = 0.2-0.3 A). The new amino acid does not coordinate to the iron, because the side chain is shorter than that of Tyr. In contrast to the wild-type enzyme, Tyr447 remains bound to the iron, as a result, a monodentate substrate complex is formed between the iron and protocatechuate 04. Protocatechuate does not shift into a chelated orientation. Inhibitors like 4-hydroybenzoate and 3-hydroybenzoate bind more tighly to the mutant enzyme, whereas the substrate protocatechuate binds less tightly. Pseudomonas putida
1.13.11.3 Y408E iron is tightly bound. The structure reveals no significant mutation-related changes except in the immediate vicinity of the altered amino acid (rmsd over all atoms = 0.2-0.3 A). The new amino acid does not coordinate to the iron, because the side chain is shorter than that of Tyr. In contrast to the wild-type enzyme, Tyr447 remains bound to the iron, as a result, a monodentate substrate complex is formed between the iron and protocatechuate 04. Protocatechuate does not shift into a chelated orientation. Pseudomonas putida
1.13.11.3 Y408F iron is not tightly bound, the Y408F mutant does not reconstitute above half-occupancy and loses color during crystallization attempts. Inhibitors like 4-hydroybenzoate and 3-hydroybenzoate bind more tighly to the mutant enzyme, whereas the substrate protocatechuate binds less tightly. Pseudomonas putida
1.13.11.3 Y408H iron is tightly bound. The structure reveals no significant mutation-related changes except in the immediate vicinity of the altered amino acid (rmsd over all atoms = 0.2-0.3 A). The new amino acid does not coordinate to the iron, because the side chain is shorter than that of Tyr. In contrast to the wild-type enzyme, Tyr447 remains bound to the iron, as a result, a monodentate substrate complex is formed between the iron and protocatechuate 04. Protocatechuate does not shift into a chelated orientation. Inhibitors like 4-hydroybenzoate and 3-hydroybenzoate bind more tighly to the mutant enzyme, whereas the substrate protocatechuate binds less tightly. Pseudomonas putida

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.13.11.3 3-hydroxybenzoate optical titration at 25°C Pseudomonas putida
1.13.11.3 4-hydroxybenzoate optical titration at 25°C Pseudomonas putida

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.13.11.3 additional information
-
additional information rates (s-1) and dissocation constants (microM) for protocatechuate: Y408H: k+2 = 2.8, k-2 = 1.9, k+2 + k-2 = 4.7, K1 = 2600, Kd = 1100, Y408F: k+2 = 0.39, k-2 = 0.46, k+2 + k-2 = 0.85, K1 = 84, Kd = 45, Y408C: k+2 + k-2 = 0.67 Pseudomonas putida
1.13.11.3 additional information
-
additional information substrate dissociation constant for protocatechuate: wild type = 2.5 microM, Y408H = 39 micro M, Y408C = 57 microM Pseudomonas putida

Organism

EC Number Organism UniProt Comment Textmining
1.13.11.3 Pseudomonas putida
-
reclassified from Pseudomonas aeruginosa
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.13.11.3 enzymes from Escherichia coli: DEAE-Sepharose Fast Flow column (5.5 x 19 cm), Phenyl-Sepharose CL-4B column (4.5 x 22.5 cm), Sephacryl S-300 column (3 x 97.5 cm) Pseudomonas putida
1.13.11.3 enzymes from Pseudomonas florescens Pseudomonas putida

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.13.11.3 3,4-dihydroxybenzoate + O2
-
Pseudomonas putida beta-carboxy-cis,cis-muconate
-
?

Synonyms

EC Number Synonyms Comment Organism
1.13.11.3 3,4-PCD
-
Pseudomonas putida

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.13.11.3 0.007
-
protocatechuate for the mutant enzyme Y408H, at 25°C in 50 mM Tris and 2 mM beta-mercaptoethanol, pH 8.5 under saturating protocatechuate and O2 conditions Pseudomonas putida
1.13.11.3 0.01
-
protocatechuate for the mutant enzyme Y408E, at 25°C in 50 mM Tris and 2 mM beta-mercaptoethanol, pH 8.5 under saturating protocatechuate and O2 conditions Pseudomonas putida
1.13.11.3 0.011
-
protocatechuate for the mutant enzyme Y408F, at 25°C in 50 mM Tris and 2 mM beta-mercaptoethanol, pH 8.5 under saturating protocatechuate and O2 conditions Pseudomonas putida
1.13.11.3 0.13
-
protocatechuate for the mutant enzyme Y408C, at 25°C in 50 mM Tris and 2 mM beta-mercaptoethanol, pH 8.5 under saturating protocatechuate and O2 conditions Pseudomonas putida
1.13.11.3 100
-
protocatechuate for the wild-type enzyme, at 25°C in 50 mM Tris and 2 mM beta-mercaptoethanol, pH 8.5 under saturating protocatechuate and O2 conditions Pseudomonas putida

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.13.11.3 8.5
-
maximum activity for the wild type enzyme and the mutants enymes Y408C, Y408F, Y408E and Y408H Pseudomonas putida

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.13.11.3 additional information
-
additional information rates (s-1) and dissociation constants (microM) for 4-hysroxybenzoate: wild type: k+2 = 42, k-2 = 14, k+2 + k-2 = 56, K1 = 700, Kd = 170, Y408H: k+2 + k-2 = 2.3, Y408C: k+2 + k-2 = 0.7, Y408F: k+2 + k-2 = 0.9 Pseudomonas putida
1.13.11.3 additional information
-
additional information substrate dissociation constant for 3-hydroxybenzoate: wild type = 3500 microM, Y408H = 180 microM, Y408E = 210 microM, Y408C = 2500 microM, Y408F = 1800 microM Pseudomonas putida
1.13.11.3 additional information
-
additional information substrate dissociation constant for 4-hydroxybenzoate: wild type = 300 microM, Y408H = 6.3 microM, Y408C = 51 microM, Y408F = 12 microM Pseudomonas putida