Unveiling the molecular architecture of Tc JHAMT : structural basis for catalytic efficiency and targeted inhibitor screening in the red flour beetle, Tribolium castaneum

BACKGROUND: The red flour beetle, Tribolium castaneum, is a holometabolous insect regulated by juvenile hormone (JH), and juvenile hormone acid methyltransferase (JHAMT) serves as a rate-limiting enzyme involved in JH biosynthesis. JHAMT thus represents a promising molecular target for pest control, although the structural properties of canonical JHAMT remain uncharacterized. RESULTS: Herein, we present the canonical JHAMT of T. castaneum, TcJHAMT3, and report its three-dimensional structure at 2.50 Å resolution. TcJHAMT3 exhibits a conserved structural pattern with insect JHAMT homologs, consisting of one Rossmann fold domain and one substrate-binding domain. Structural and biochemical analyses revealed that the insertion of four to six amino acids and the formation of the β7' strand in TcJHAMT3 constitute critical structural determinants underlying the methyltransferase activity of canonical JHAMTs. Furthermore, a novel JHAMT-targeting inhibitor, JI3, was identified through structure-based screening and inhibitory analyses, and it can effectively disrupt the development and metamorphosis processes of T. castaneum. CONCLUSION: Structural and biochemical analyses enabled the definition of the molecular basis for understanding the catalytic activity of canonical JHAMT. Additionally, the findings from this work provide a blueprint for the development of more potent specific inhibitors of JHAMT and present a novel lead compound for management and control of T. castaneum. © 2025 Society of Chemical Industry.