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 |
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 |
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 |
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 |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.13.11.3 | Pseudomonas putida | - |
reclassified from Pseudomonas aeruginosa | - |
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 |
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 | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.13.11.3 | 3,4-PCD | - |
Pseudomonas putida |
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 |
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 |
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 |