Insights into tandem dioxygenases—copper radical oxidases in fungal Phytopathogens of the Colletotrichum acutatum species complex

Fungal phytopathogens represent a major threat to global agriculture, causing extensive yield losses. Among them, Colletotrichum spp. are infamous for their capacity of infecting a wide variety of monocot and dicot hosts. Motivated by the recent discovery in Colletotrichum orbiculare of a redox relay between Copper Radical Alcohol Oxidases from the AA5_2 subfamily and tandem peroxidases involved in plant infection, we investigated species from the Colletotrichum species complex ( C. acutatum and C. tamarilloi ) where the tandem peroxidase gene is absent. Instead, putative ring‐cleavage dioxygenases are found within a conserved genomic locus adjacent to AA5_2 paralogues. Therefore, we hypothesized that ring‐cleavage dioxygenases could function as redox partners for AA5_2. Given the limited information available on fungal dioxygenases, we performed a biochemical characterization of both Colletotrichum ring‐cleavage dioxygenases. LC–MS analyses revealed that both enzymes catalyze intradiol (1,2‐) ring cleavage of catecholic substrates and preferentially oxidize methyl‐substituted catechols. Structural modeling indicated an open, solvent‐exposed active site resembling that of spider mite intradiol ring cleavage dioxygenases and distinct from the compact, oligomeric bacterial homologs. However, both dioxygenases showed only a limited capacity to activate AA5_2 in vitro, indicating that close genomic proximity does not necessarily imply efficient functional coupling. Together, these biochemical insights into both AA5_2 and ring‐cleavage dioxygenases provide a foundation for future studies aimed at elucidating their biological roles in fungal metabolism and pathogenicity.