Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa

Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry. Barsottini et al. characterize the alternative oxidase (MpAOX), which is crucial for the survival of Moniliophthora perniciosa, a fungal pathogen of cocoa. They identify key residues for the interactions between this enzyme and its inhibitors, guiding the future development of AOX-targeting antifungal agents that would benefit the chocolate industry. (AU)