1.1.3.38 C470L mutant displays similar activity to the wild-type enzyme with the substrates vanillyl alcohol, chavicol aund eugenol, but no activity with linear 4-alkylphenols 763480 1.1.3.38 D170A 3100fold decrease in turnover-number for for 4-(methoxymethyl)phenol 655977 1.1.3.38 D170A only 50% of the FAD is covalently bound. With vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is more than 1000fold less active than the wild-type enzyme 654161 1.1.3.38 D170A/T457E produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol 654161 1.1.3.38 D170E 2fold increase in KM-value for for 4-(methoxymethyl)phenol, 2.1fold increase in Km-value for vanillyl alcohol, 2fold decrease in KM-value for eugenol as compared to wild-type enzyme.442fold decrease in turnover-number for for 4-(methoxymethyl)phenol, 2.5fold decrease in turnover-number for vanillyl alcohol, 51.5fold decrease in turnover-number for eugenol as compared to wild-type enzyme.Redox potential of mutant enzyme, + 6 mV, is decreased compared to wild-type enzyme, 55 mV 655977 1.1.3.38 D170E substrate preference is similar to wild-type enzyme, as the wild-type enzyme the mutant enzyme favors the production of alkenes 655540 1.1.3.38 D170E with vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is 5-fold to 100fold less active than the wild-type enzyme 654161 1.1.3.38 D170N 1550fold decrease in turnover-number for for 4-(methoxymethyl)phenol 655977 1.1.3.38 D170N no FAD is covalently bound. With vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is more than 1000fold less active than the wild-type enzyme 654161 1.1.3.38 D170S 2fold decrease in KM-value for eugenol as compared to wild-type enzyme.1290fold decrease in turnover-number for for 4-(methoxymethyl)phenol, 825fold decrease in turnover-number for vanillyl alcohol, 1750fold decrease in turnover-number for eugenol as compared to wild-type enzyme. Redox potential of mutant enzyme, -91 mV, is decreased compared to wild-type enzyme, 55 mV 655977 1.1.3.38 D170S most active with branched-chain 4-alkylphenol, mutant enzyme favors the formation of alcohols 655540 1.1.3.38 D170S with vanilly alcohol, eugenol, and 4-(methoxymethyl)phenol the mutant enzyme is more than 1000fold less active than the wild-type enzyme 654161 1.1.3.38 D170S/T457E produces (S)-1-(4'-hydroxyphenyl)ethanol from 4-ethylphenol. The wild-type enzyme produces (R)-1-(4'-hydroxyphenyl)ethanol 654161 1.1.3.38 E502G the octamer/dimer ratio is 1:10. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols 656232 1.1.3.38 F424G mutant does not contain any flavin after purification 763480 1.1.3.38 F454Y as for wild-type enzyme the octamer/dimer ratio of the mutant enzyme is 1.5:1. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols 656232 1.1.3.38 H422A mutant enzyme retains activity, turnover rates decrease by 1 order of magnitude. Mutant enzyme is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. Although mutation prevents covalent linkage of FAD, mutant enzyme contains tightly bound FAD 655975 1.1.3.38 H422C mutant enzyme retains activity, turnover rates decrease by 1 order of magnitude. Although mutation prevents covalent linkage of FAD, mutant enzyme contains tightly bound FAD 655975 1.1.3.38 H422T mutant enzyme retains activity, turnover rates decrease by 1 order of magnitude. Although mutation prevents covalent linkage of FAD, mutant enzyme contains tightly bound FAD 655975 1.1.3.38 H61T FAD-free apoenzyme H61T mainly exists as a dimeric species of 126000 Da. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization 656076 1.1.3.38 H61T in the mutant enzyme the covalent His-C8alpha-flavin linkage is not formed, while the enzyme is still able to bind FAD and perform catalysis. The mutant enzyme is about 10fold less active with 4-(methoxymethyl)phenol than the wild-type enzyme. Crystal structure of both the holo and apo form of H61T are highly similar to the structure of the wild-type enzyme 655990 1.1.3.38 I238T the octamer/dimer ratio is 4:1. The catalytic efficiency of the mutant is significantly increased for ortho-substituted 4-methylphenols 656232 1.1.3.38 I468V mutant displays similar activity to the wild-type enzyme with the substrates vanillyl alcohol, chavicol aund eugenol, but no activity with linear 4-alkylphenols 763480 1.1.3.38 L316M mutant displays substrate specificity profile similar to wild-type 763480 1.1.3.38 additional information exchange of a loop at the dimer-dimer interface in octameric vanillin oxidase that is not present in dimeric EUGO. A vanillin oxidase variant where the loop was deleted, loopless VAO, exclusively forms dimers. Introduction of the loop into EUGO is not sufficient to induce its octamerization. Neither variant displays major changes in its catalytic properties as compared to the wild-type enzyme 763012 1.1.3.38 T457Q mutant shows about 3fold increased activity towards vanillyl alcohol, but decrease in activity with all other substrates 763480 1.1.3.38 T459I mutant displays substrate specificity profile similar to wild-type 763480 1.1.3.38 T505S as for wild-type enzyme the octamer/dimer ratio of the mutant enzyme is 1.5:1 656232 1.1.3.38 Y108F deprotonation of the substrate's phenol group is impaired 763252 1.1.3.38 Y108F/Y503F deprotonation of the substrate's phenol group is impaired 763252 1.1.3.38 Y503F deprotonation of the substrate's phenol group is impaired 763252