Structural insights into ortho-aminophenol oxidases: kinetic and crystallographic characterization of SmNspF and SgGriF

Actinobacteria-derived o-aminophenol oxidases (AOs) represent a largely unexplored subclass of type-III copper enzymes with catalytic properties distinct from tyrosinases and catechol oxidases. The determination of the first crystal structure of an AO (SmNspF) displays unique loop insertions and important second-sphere amino acids in vicinity of the binuclear copper center. The substrate-guiding effect of the second activity controller (His_B2+1) influences the binding affinity for carboxyl-containing substrates in the AOs SmNspF and SgGriF. Thus, kinetic investigations reveal both overlapping and distinct substrate preferences for SmNspF and SgGriF: while both enzymes oxidize monophenols, o-aminophenols, and o-diphenols, they do so at significantly different reaction rates. SmNspF preferentially oxidizes carboxylated substrates such as 3,4-dihydroxybenzoic acid and 3-amino-4-hydroxybenzoic acid, whereas SgGriF exhibits higher activity toward para-methylated analogs, including 4-methylcatechol and 2-amino-4-methylphenol. Remarkably, both enzymes display enzymatic activities beyond the known AO reactivity spectrum by oxidizing 2-aminoresorcinol and o-phenylenediamine, which underlies the high versatility of the binuclear copper center. Altogether, these findings provide a structural basis for AO's enzymatic activity and broaden the known catalytic spectrum, which enables the prediction of catalytic properties in type-III copper proteins based on their amino acid sequence.