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Literature summary for 1.7.1.6 extracted from

  • Brissos, V.; Goncalves, N.; Melo, E.; Martins, L.
    Improving kinetic or thermodynamic stability of an azoreductase by directed evolution (2014), PLoS ONE, 9, e87209 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression in Escherichia coli strains Tuner(DE3) and KRX Pseudomonas putida

Protein Variants

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 [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information wild-type and mutant kinetics and thermodynamics, overview Pseudomonas putida

Organism

Organism UniProt Comment Textmining
Pseudomonas putida
-
-
-
Pseudomonas putida MET94
-
-
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the bifunctional enzyme shows activity with quinone substrates, anthraquinone-2-sulfonic acid, 1,4-benzoquinone, 1,2-dihydroxybenzene (catechol), 2-hydroxy-1,4-naphthoquinone (Lawsone), and 1,2-naphthoquinone-4-sulfonate, and NADPH as cofactor, cf. EC 1.6.5.10 Pseudomonas putida ?
-
?

Subunits

Subunits Comment Organism
homodimer
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Pseudomonas putida

Synonyms

Synonyms Comment Organism
azoreductase
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Pseudomonas putida
PpAzoR
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Pseudomonas putida

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Pseudomonas putida

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
assay at Pseudomonas putida

Cofactor

Cofactor Comment Organism Structure
NAD(P)H
-
Pseudomonas putida

General Information

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