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Literature summary for 1.1.3.7 extracted from

  • Vina-Gonzalez, J.; Gonzalez-Perez, D.; Ferreira, P.; Martinez, A.T.; Alcalde, M.
    Focused directed evolution of aryl-alcohol oxidase in Saccharomyces cerevisiae by using chimeric signal peptides (2015), Appl. Environ. Microbiol., 81, 6451-6462 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.1.3.7

Cloned(Commentary)

Cloned (Comment) Organism
gene aao, recombinant expression of wild-type and mutant enzymes in Saccharomyces cerevisiae Pleurotus eryngii

Protein Variants

Protein Variants Comment Organism
H91N random mutageneis, the FX7 mutant (harboring the H91N mutation) shows a dramatic 96fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirms the correct processing of the prealphaproK hybrid peptide by the KEX2 protease. FX7 shows higher stability in terms of pH and temperature, whereas the pH activity profiles and the kinetic parameters are maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for Pleurotus eryngii and Pleurotus pulmonarius AAO. FX7 mutant homology modeling using the crystal structure of the AAO from Pleurotus eryngii at a resolution of 2.55 A, PDB ID 3FIM, structure-function analysis Pleurotus eryngii
additional information in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promotes AAO expression and stability. The native signal sequence of AAO from Pleurotus eryngii is replaced by those of the mating alpha-factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae strain BJ5465. The secretion of these mutant AAO constructs increase in the following descending order: preproalpha-AAO, prealphaproK-AAO, preKproalpha-AAO, preproK-AAO. The chimeric prealphaproK-AAO is subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination is concentrated on two protein segments (Met[alpha1]-Val109 and Phe392-Gln566), and an array of improved variants is identified Pleurotus eryngii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Pleurotus eryngii
0.034
-
 4-methoxybenzyl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
0.035
-
 4-methoxybenzyl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
0.059
-
 2,4-hexadien-1-ol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
0.087
-
 2,4-hexadien-1-ol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
0.388
-
 benzyl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
0.504
-
 benzyl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
0.51
-
 veratryl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
0.77
-
 veratryl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii

Localization

Localization Comment Organism GeneOntology No. Textmining
extracellular
-
 Pleurotus eryngii
-
-

Organism

Organism UniProt Comment Textmining
Pleurotus eryngii
-
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
24
-
 purified recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
74
-
 purified recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,4-hexadien-1-ol + O2
-
 Pleurotus eryngii 2,4-hexandienal + H2O2
-
 ?
4-methoxybenzyl alcohol + O2
-
 Pleurotus eryngii 4-methoxybenzaldehyde + H2O2
-
 ?
benzyl alcohol + O2
-
 Pleurotus eryngii benzaldehyde + H2O2
-
 ?
additional information the enzyme shows a broad substrate specificity and highly stereoselective reaction mechanism. Assay method using ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)]/horseradish peroxidase Pleurotus eryngii ?
-
 ?
veratryl alcohol + O2
-
 Pleurotus eryngii veratrylaldehyde + H2O2
-
 ?

Subunits

Subunits Comment Organism
? x * 61485, recombinant enzyme mutant H91N, mass spectrometry, x * 61847, recombinant wild-type enzyme, mass spectrometry, x * 61088, recombinant wild-type enzyme, sequence calculation, x * recombinant enzyme mutant H91N, sequence calculation, x * 65000, recombinant wild-type enzyme, SDS-PAGE, x * 120000, recombinant enzyme mutant H91N, SDS-PAGE Pleurotus eryngii

Synonyms

Synonyms Comment Organism
AAO
-
 Pleurotus eryngii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
24
-
 assay at Pleurotus eryngii

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
47.5
-
 T50 of recombinant wild-type enzyme Pleurotus eryngii
64.3
-
 T50 of recombinant mutant H91N Pleurotus eryngii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2 8 veratryl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
19
-
 benzyl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
22
-
 benzyl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
52
-
 2,4-hexadien-1-ol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
54
-
 4-methoxybenzyl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
66
-
 veratryl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
105
-
 4-methoxybenzyl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
136
-
 2,4-hexadien-1-ol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5
-
 wild-type and mutant enzymes Pleurotus eryngii

Cofactor

Cofactor Comment Organism Structure
FAD
-
 Pleurotus eryngii

pI Value

Organism Comment pI Value Maximum pI Value
Pleurotus eryngii recombinant wild-type enzyme, isoelectric focusing
-
 3.9
Pleurotus eryngii recombinant H91N FX7 mutant, isoelectric focusing
-
 4.3

General Information

General Information Comment Organism
additional information residue 91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily as Asn91, except for Pleurotus eryngii and Pleurotus pulmonarius AAO Pleurotus eryngii
physiological function aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay Pleurotus eryngii

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
28
-
 veratryl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
36
-
 veratryl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
71
-
 benzyl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
131
-
 benzyl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
866
-
 2,4-hexadien-1-ol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
1555
-
 2,4-hexadien-1-ol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii
1562
-
 4-methoxybenzyl alcohol recombinant H91N FX7 mutant, pH 6.0, 24°C Pleurotus eryngii
2979
-
 4-methoxybenzyl alcohol recombinant wild-type enzyme, pH 6.0, 24°C Pleurotus eryngii