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

Mario R O Barsottini,Gonçalo A G Pereira,Anthony L Moore,Alice Copsey,Renata M Baroni,Luke Young,Artur T Cordeiro

doi:10.1038/s42003-020-0981-6

Abstract

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.

Highlights

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 cocoaplanting countries
The recombinant MpAOX (rMpAOX) construct used in this work corresponds to the mature M. perniciosa AOX (MpAOX) enzyme without the predicted mitochondrial leading peptide, which is cleaved following import into the mitochondrial matrix[39]
A dose–response assay with AMP, ADP and GMP reveals that AMP activates rMpAOX as much as GMP at higher concentrations, whereas 6.5 mM ADP was not enough to sustain maximum rMpAOX rates

Summary

Introduction

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 cocoaplanting countries. We 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. The basidiomycete fungus Moniliophthora perniciosa is the causal agent of the witches’ broom disease of cocoa (WBD) in Latin America, and a WBD outbreak in Brazil’s main cocoaproducing region—the state of Bahia—in 1989 led to a substantial loss in Brazilian competitiveness. We have shown that AOX is highly expressed during the M. perniciosa–cocoa interaction and is critical for disease progression[4,5]

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