Abstract
<jats:p> The generalist <jats:italic>Helicoverpa armigera</jats:italic> and the specialist <jats:italic>Helicoverpa assulta</jats:italic> are closely related noctuid pests and are among the few insect herbivores capable of feeding on and damaging hot pepper fruits, which contain the defensive compound capsaicin. Cytochrome P450 monooxygenases (P450s) contribute to the metabolism of plant defensive compounds and can facilitate insect adaptation to chemically defended host plants. Here, we identified <jats:italic>CYP6AN1</jats:italic> in <jats:italic>H. assulta</jats:italic> ( <jats:italic>HassCYP6AN1</jats:italic> ) and comparatively characterized the <jats:italic>CYP6AN1</jats:italic> orthologs from <jats:italic>H. armigera</jats:italic> ( <jats:italic>HarmCYP6AN1</jats:italic> ) and <jats:italic>H. assulta</jats:italic> . RACE identified one full-length <jats:italic>HarmCYP6AN1</jats:italic> transcript and three full-length <jats:italic>HassCYP6AN1</jats:italic> transcript isoforms. The <jats:italic>HassCYP6AN1</jats:italic> isoforms contained distinct 5′ UTRs generated by alternative transcription initiation and splicing but shared an identical coding sequence. Sequence comparisons and phylogenetic analysis supported their assignment as orthologs. Constitutive <jats:italic>CYP6AN1</jats:italic> expression was higher in the <jats:italic>H. assulta</jats:italic> midgut, whereas dietary capsaicin significantly induced <jats:italic>CYP6AN1</jats:italic> expression in the <jats:italic>H. armigera</jats:italic> midgut. Recombinant CYP6AN1 proteins were co-expressed with NADPH-cytochrome P450 reductase in <jats:italic>Escherichia coli</jats:italic> , and their substrate-metabolizing activities were evaluated using HPLC-based depletion assays. Both orthologs metabolized capsaicin, but HarmCYP6AN1 exhibited an approximately 2.3-fold higher depletion activity than HassCYP6AN1 under the conditions tested. HarmCYP6AN1 also showed P450-content-dependent xanthotoxin depletion, whereas no detectable xanthotoxin metabolism was observed for HassCYP6AN1. These findings establish CYP6AN1 as a component of the capsaicin-metabolizing repertoire of both species and reveal substantial divergence between the orthologs in transcript organization, expression regulation, catalytic activity, and detectable substrate range. </jats:p>