Cloned (Comment) | Organism |
---|---|
recombinant expression in Escherichia coli strains Tuner(DE3) and KRX | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
additional information | improvement of the kinetic and thermodynamic stability of the azoreductase by directed evolution via rational design approaches, five rounds of mutagenesis/recombination are followed by high-throughput screening. Mutant 1B6 shows a 300fold higher half-life at 50°C compared to the wild-type enzyme. mutant 1B6 has a folded state slightly less stable than the wild-type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant is therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturb hydrophobic patches and increase the surface net charge of the protein. Mutants 2A1 and 2A1-Y179H show increased thermodynamic stability with a 10-20°C higher melting temperature than wild-type, these residues are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state. Molecular details of mutations that improve stability, overview | Pseudomonas putida |
Q192R | site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 60 min) relative to parental mutant variant B1G6 | Pseudomonas putida |
Q192R/A46P/V159A | site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 90 min) relative to parental mutant variant 16B7 | Pseudomonas putida |
Q192R/A46P/V159A/A48P | site-directed mutagenesis, analysis of initial activity and thermostability (at 60°C, 45 min) relative to parental mutant variant 2A1 | Pseudomonas putida |
Q192R/A46P/V159A/C129S | site-directed mutagenesis, analysis of initial activity and thermostability (at 60°C, 45 min) relative to parental mutant variant 23C10 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/A178D/A31S/K74E/A88G/L143Q | site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 2F11 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/A178D/A77T/F98L/N131D | site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 3B9 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/A178D/A88G/N131D/L143Q | site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 1B6 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/A178D/K74E/L143Q | site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 2E4 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/A178D/N131D/L143Q | site-directed mutagenesis, analysis of initial activity and thermostability (at 85°C, 150 min) relative to parental mutant variant 6F11 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/A77T/N131D | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 14D4 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/D7H/A178D | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 13G10 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/E36D/L143Q | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 1C11 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/I6V/T79R/Y179H | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 32F5 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/K74E/A88G | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 23C5 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/L161M/L169P | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 27E4 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/N14D/L143Q | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 6F10 | Pseudomonas putida |
Q192R/A46P/V159A/C129S/Y179H | site-directed mutagenesis, analysis of initial activity and thermostability (at 80°C, 60 min) relative to parental mutant variant 23E4 | Pseudomonas putida |
Q192R/A46P/V159A/Y179H | site-directed mutagenesis, analysis of initial activity and thermostability (at 60°C, 45 min) relative to parental mutant variant 19E4 | Pseudomonas putida |
Q192R/Y179H | site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 90 min) relative to parental mutant variant 12B8 | Pseudomonas putida |
Y179H | site-directed mutagenesis, analysis of initial activity and thermostability (at 55°C, 60 min) relative to parental mutant variant K7E3 | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | wild-type and mutant kinetics and thermodynamics, overview | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | - |
- |
- |
Pseudomonas putida MET94 | - |
- |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | the bifunctional enzyme also shows activity with azo dyes and NAD(P)H as cofactor, cf. EC 1.7.1.6 | Pseudomonas putida | ? | - |
? | |
additional information | the bifunctional enzyme also shows activity with azo dyes and NAD(P)H as cofactor, cf. EC 1.7.1.6 | Pseudomonas putida MET94 | ? | - |
? | |
NADPH + H+ + 1,2-naphthoquinone-4-sulfonate | i.e. Lawsone | Pseudomonas putida | NADP+ + ? | - |
? | |
NADPH + H+ + 1,2-naphthoquinone-4-sulfonate | i.e. Lawsone | Pseudomonas putida MET94 | NADP+ + ? | - |
? | |
NADPH + H+ + 1,4-benzoquinone | - |
Pseudomonas putida | NADP+ + ? | - |
? | |
NADPH + H+ + 1,4-benzoquinone | - |
Pseudomonas putida MET94 | NADP+ + ? | - |
? | |
NADPH + H+ + 2-hydroxy-1,4-naphthoquinone | - |
Pseudomonas putida | NADP+ + ? | - |
? | |
NADPH + H+ + 2-hydroxy-1,4-naphthoquinone | - |
Pseudomonas putida MET94 | NADP+ + ? | - |
? | |
NADPH + H+ + a quinone | - |
Pseudomonas putida | NADP+ + a quinol | - |
? | |
NADPH + H+ + a quinone | - |
Pseudomonas putida MET94 | NADP+ + a quinol | - |
? | |
NADPH + H+ + anthraquinone-2-sulfonic acid | - |
Pseudomonas putida | NADP+ + ? | - |
? | |
NADPH + H+ + catechol | i.e. 1,2-dihydroxybenzene | Pseudomonas putida | NADP+ + ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | - |
Pseudomonas putida |
Synonyms | Comment | Organism |
---|---|---|
azoreductase | - |
Pseudomonas putida |
PpAzoR | - |
Pseudomonas putida |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Pseudomonas putida |
General Information | Comment | Organism |
---|---|---|
physiological function | Pseudomonas putida MET94 is a bacteria that degrades a wide range of structurally distinct azo dyes with high efficiency and the azoreductase PpAzoR plays a key role in this process | Pseudomonas putida |