Increased energy, materials, and food production driven by rising populations has resulted in society’s consumption habits exceeding resource availability. Thus, there is a need for sustainable processes and towards this, enzyme biocatalysis holds significant potential. Particularly, Copper Radical Oxidases (CROs) from Auxiliary Activity Family 5 (AA5) in the Carbohydrate-Active Enzyme (CAZy) classification are attractive candidates for their diverse biotechnological potential in alcohol and aldehyde transformation, and their roles in fungal development and phytopathogenesis. CROs include galactose-6-oxidases and glyoxal oxidases, which were first characterised in 1959 and 1987, respectively. Within AA5, glyoxal oxidases and galactose-6-oxidases are classified into subfamilies 1 and 2 (AA5₁, AA5₂), respectively. Given the surge in fungal, bacterial, and plant genome sequences, molecular phylogeny has indicated that CRO diversity remains largely underexplored. While CRO research has centred on the biocatalytic potential of AA5₂ members, much less is known about AA5₁ and the biological roles of CROs. Hence, the overarching goal of this thesis is to provide new insight into CRO function through combined biochemical, structural, and biological studies, with a focus on fungal members. This work presents the further exploration of both AA5 subfamilies. Detailed bioinformatic and biochemical analyses of six new AA5₂ enzymes show that CRO activities are not monophyletic, and the presence of a key tryptophan/arginine pair as a likely indicator of galactose-6-oxidase activity is further validated. Furthermore, the first crystal structure of an AA5₁ enzyme is presented and comparisons with AA5₂ members reveal that the distinctive activities between subfamilies may arise from differences in spin density distributions within the active site second coordination sphere. Lastly, due to similarities with CRO gene organisation in Colletotrichum fungi, CROs from two Verticillium species were investigated. Biochemical and reverse genetics studies of CROs from Verticillium dahliae suggest that the biological roles of CROs are not conserved across fungi. The combinatorial approaches employed within this thesis expands our understanding of fundamental structure-function relationships behind the diverse activities in the AA5 family and the roles CROs play in phytopathogenic fungi. This work can be used to inform future studies regarding CRO engineering for biocatalysis or methods in combatting plant diseases.
Jessica Kalyun Fong (Thu,) studied this question.