Mechanistic and biocatalytic enzymology of flavoprotein oxidases

Flavoprotein oxidases catalyse the two-electron oxidation of their substrates using molecular oxygen as an electron acceptor. This makes them interesting candidates for biocatalytic applications. Many flavin-dependent oxidases belong to one of a number of families of structurally related proteins. One such family is the VAO/PCMH family, which contains enzymes that share a common FAD-binding fold. Within this family, a subfamily of enzymes is found that catalyse the oxidation of para-substituted phenols, the 4-phenol oxidising (4PO) subfamily. Enzymes from this subfamily include the oxidases vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) and the dehydrogenase para-cresol methylhydroxylase (PCMH). This thesis focusses on understanding the mode of action of these enzymes and exploring their potential for biocatalytic applications. Although the main focus is on the 4PO subfamily, other flavin-dependent enzymes are also discussed at some points. Broadly speaking, the thesis consists of two parts. In the first, the emphasis is on understanding the mode of action of 4PO subfamily members at the molecular level. Chapter 2 provides an overview of the current state of our knowledge on members of the VAO/PCMH family. The family was divided into eleven subfamilies based on a phylogenetic analysis of 115 members. These subfamilies are discussed in terms of the reactions they catalyse and our mechanistic understanding thereof. Chapter 3 explores the differing oligomerisation states of VAO and EUGO. Despite their highly similar secondary and tertiary structures, VAO forms octamers, which can be described as tetramers of stable dimers, whereas EUGO is exclusively dimeric. A single loop, which is located at the dimer-dimer interface of a VAO octamer, was shown to be essential for the octamerisation of VAO. Its deletion led to a VAO variant that is exclusively dimeric in solution and displays similar catalytic properties to the wild-type enzyme. Chapter 4 focusses on understanding the mechanism of substrate activation in VAO. To enable catalysis, the substrate must first be bound in the active site in its phenolate form. The role of two tyrosine residues, Tyr-108 and Tyr-503, in facilitating substrate deprotonation was studied by site-directed mutagenesis. These tyrosine residues play a crucial role in enabling substrate activation by VAO, both by stimulating preferential binding of the phenolate form of the substrate and by deprotonating the substrate when it is bound in its protonated form. The second part of the thesis focusses on the biocatalytic applications of flavin-dependent oxidases. Chapter 5 provides an overview of how flavin-dependent oxidases can be used in synthetic organic chemistry. Chapter 6 describes the development of a high-throughput screening assay for the substrate specificity of variants of VAO and EUGO based on the detection of hydrogen peroxide formed by the enzymes using a xylenol orange assay. This assay was used to screen the substrate specificity of the wild-type enzymes and a small library of variants, leading to the identification of novel substrates of the enzymes and two variants with improved activity towards at least one substrate. Chapter 7 describes the scale-up of the synthesis of a chiral secondary alcohol, (R)-1-(4′-hydroxyphenyl)ethanol, using VAO. Under optimised reaction conditions, we obtained 4.1 g of pure (R)-1-(4′-hydroxyphenyl)ethanol. In Chapter 8, the focus moves away from the 4PO subfamily. It describes the oxidation of thiols by a number of flavin-dependent alcohol oxidases. Alditol oxidase (AldO) and 5-(hydroxymethyl)furfural oxidase (HMFO) were shown to oxidise thiol analogues of their alcohol substrates to the corresponding thiocarbonyls, which have a number of potential applications in organic synthesis. These results provide advances in our understanding of the mode of action and potential applications of flavoprotein oxidases and will facilitate their future application in industrial biocatalytic processes.