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

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 cocoaplanting 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.

Kinetic characterisation and inhibitor sensitivity of Candida albicans and Candida auris recombinant AOX expressed in a self-assembled proteoliposome system

“…However, it should be noted that there is a significant decrease in efficacy of the compounds with the fungal AOX’s when compared to TAO, requiring a 250 fold increase in concentration. These values are reasonably consistent with those seen against other fungal AOX’s 50 , which has largely been attributed to the decrease in size of the hydrophobic cavity in the fungal species when compared to TAO being problematic for inhibitor egress 42 , 51 .…”

“…PLs were prepared exactly as described in previously published protocols using phospholipids; phosphatidylcholine, phosphatidylethanolamine and cardiolipin, mixed at a ratio of 8:1:1 25 , 29 . A typical preparation used a total of 10 mg phospholipids and 100 nmol of Q 10 , both of which are essential for PL activity 50 . The only modifications to the protocol were the detergent used to partially solubilise the phospholipid/Q 10 mixture (0.5% DDM in our preparations) and the protein content of the phospholipid vesicles as follows.…”

Haloadaptative Responses of Aspergillus sydowii to Extreme Water Deprivation: Morphology, Compatible Solutes, and Oxidative Stress at NaCl Saturation

“…The activity of alternative oxidases is crucial when occurs an accumulation of complex III-derived O 2 − as a consequence of the oxidative stress [ 47 ]. It is known that aox genes are linked to fungal development under stress conditions [ 75 , 76 ]. In Candida albicans , alternative oxidases are intimately involved in the yeast growth during exposure to fluconazole [ 77 ], while these enzymes are relevant during the growth of A. fumigatus [ 78 , 79 ] and Botrytis cinerea [ 76 ] under hypoxic conditions.…”

Comparison of the Kinetic Parameters of Alternative Oxidases From Trypanosoma brucei and Arabidopsis thaliana—A Tale of Two Cavities

“…For instance, plant AOXs are activated by organic acids (Millar et al, 1993;Umbach et al, 2002;Selinski et al, 2018). In contrast, the AOX in fungi and protists is generally activated by mono-and diphosphate nucleosides, such as adenosine monophosphate (AMP), guanosine monophosphate (GMP), and adenosine diphosphate (ADP) (Jarmuszkiewicz et al, 2005;Barsottini et al, 2020). Those allosteric activation sites have been traced to unique features in their primary structure, such as an exclusively conserved cysteine in plant AOXs (Umbach and Siedow, 2000;Umbach et al, 2006).…”