A Pair of FAD-Dependent Carbohydrate Oxidases from the Corn Smut Pathogen Ustilago maydis Oxidize Chitosan Oligomers

Auxiliary activity family 7 (AA7) oxidoreductases are fungal flavoenzymes that catalyze the C1 oxidation of diverse oligosaccharides coupled to the reduction of molecular oxygen (oxidase activity) or organic molecules (dehydrogenase activity). These enzymes are predominantly derived from Ascomycota, with a smaller, less-explored fraction from Basidiomycota and Oomycota plant pathogens. Moreover, AA7 members are promising biocatalysts for the selective oxidation of carbohydrates, which is an enduring challenge in catalysis. However, the sequence space of AA7 enzymes remains largely uncharted, particularly within basidiomycota members, and the molecular determinants shaping substrate selectivity remain ill-defined. Focusing on the basidiomycete maize pathogen Ustilago maydis, we explored the boundaries of the AA7 sequence space by identifying and characterizing two enzymes, UmAA7A and UmAA7B, that are divergent from hitherto described members. We found that UmAA7s oxidize both chitooligosaccharides (CHOS) and their deacetylated forms (dCHOS) in an acetylation-site-dependent manner. The X-ray crystal structure of UmAA7A, sequence, and structural comparisons with a model of UmAA7B and other known CHOS-active AA7s, combined with docking analyses on CHOS and dCHOS, revealed specific active-site residues behind this unprecedented substrate specificity. Moreover, a previously not reported combination of a bicovalently tethered FAD and an atypical arrangement of residues at re-side of the FAD cofactor was associated with a mainly dehydrogenase activity profile, contrasting the majority of oxidases in AA7. Altogether, this work reveals the enzymatic oxidation of dCHOS, extending the substrate scope of AA7s and laying the biochemical foundation for uncovering their biological functions during plant infection.