Vanillyl alcohol oxidases (VAOs) are flavin-dependent oxidases, belonging to the VAO/PCMH flavoprotein family and are found in ascomycetous fungi, where they are involved in the natural degradation of lignin. VAOs perform oxidation reactions on p-substituted phenols, including oxidations of alcohols, hydroxylation reactions, dehydrogenation reactions, deamination reactions and oxidative ether cleavages. To date, two VAOs have been identified, from Penicillin simplicissimum (PsVAO), and from Diplodia corticola (DcVAO). DcVAO was identified by a phylogenetic comparison of the subgroup of the 4-phenol oxidases from the VAO/PCMH flavoprotein family. For this, focus was put on the comparison of the active sites, which has been elucidated for PsVAO. VAOs harbour a relatively conserved active site however, there are differences, which are mainly found in regions, responsible for interactions with the substrate molecule. DcVAO stood out as it harbours an exchange of two amino acids at positions 424 and 470 (PsVAO numbering), where a Phe and a Cys are exchanged by an Ala and a Glu. We characterized DcVAO and found that it has the same reaction pattern as PsVAO however, DcVAO can convert 2,6-dimethoxy substituted phenols, while PsVAO cannot. In addition, DcVAO also performed better in oxidative ether cleavages. We suspected that the gained space by the Phe Ala exchange allows for the conversion of 2,6-dimethoxy substituted phenols, while the Glu may enhance ether cleavages. It could be shown that the introduction of these two is allowing for the correct positioning of 2,6-dimethoxy substituted phenols, as an exchange of the Glu by Cys resulted in similar activities on ethers, while hydroxylation reactions were greatly reduced. In silico experiments showed that in DcVAO, that substrates are differently oriented compared to PsVAO, which seems to be needed to accommodate 2,6-dimethoxy substituted phenols. This would make DcVAO a good candidate for a biocatalyst for the valorisation of lignin sourced from hardwood, as this contains almost exclusively 2,6-dimethoxylated phenolic building blocks. The initial characterization of DcVAO however also indicated that in its wildtype form is not that applicable for biocatalysis.
To drive DcVAO towards a more applicable biocatalyst, we set out to perform rational design to enhance its thermostability, influence its stereoselectivity and to optimize its reaction conditions.