Hidden treasures of bacterial eugenol oxidase flavoprotein family: versatile biocatalysts

• Applicant: Professor Dr. Dirk Tischler
• Subject Area: Biochemistry ; Biological Process Engineering
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 538360990

Flavins, especially flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are ubiquitous cofactors found among the tree of life. Due to their defined redox properties, they support proteins and turn them into powerful catalysts performing complex reactions. The latter are mostly redox reactions during which the redox state of the flavins is altered. This goes along with changes in the spectral properties of the holo-enzymes and, to be more precise, with the cofactor itself. Hence, the well-defined redox states of flavins can be used to study enzyme kinetics to uncover their reaction mechanism. In this proposal, we will make use of a just solved crystal structure of a novel bacterial FAD-dependent eugenol oxidase designated as SspEUGO. A phylogenetic study, homology modelling and molecular dynamics prompted us to investigate this enzyme as it was supposed to be more tolerant towards substrates as it comprised a more open active site compared to so far investigated enzymes. The prototype of this FAD-dependent enzyme class is vanillyl alcohol oxidase (VAO) which is found in the designation as well; VAO-family (EC 1.1.3.38). Respective enzymes harbor a covalently bound FAD cofactor relevant for substrate oxidation. This enzyme is long known and well-studied with respect to biochemistry and biocatalysis. Originally obtained from the fungus Penicillium simplicissimum, VAO is unfortunately difficult to produce recombinantly. Hence bacterial counterparts were searched of which only two were described to some extent, so far. These are eugenol oxidase (EUGO) from Rhodococcus jostii and 4-ethylphenol oxidase (Gc4EO) from Gulosibacter chungangensis. Those oxidases accept phenol-like substrates, such as vanillyl alcohol or eugenol. These are used to reduce the FAD and while doing so a para-quinone methide intermediate is formed which turns into an oxidized product in dependence on the nature of the substrate. Then, reduced FAD is recycled by molecular oxygen to yield hydrogen peroxide as a second product. However, there are remaining questions on the catalytic cycle such as 1) pH-dependency of reductive and oxidative half-reactions, 2) substrate activation by a catalytic triad and its flexibility, 3) oxygen reaction as rate-limitation, and 4) which FAD species occur during catalysis. Bacterial VAO-like oxidases including SspEUGO can easily be produced with high yields and fully flavinylated. Thus, they can function as model enzymes to study the mechanism in more detail. The knowledge on the structure-function relation will further allow to engineer these and related enzymes.

• DFG Programme: Research Grants