From structure to function: conformational dynamics and differential binding of substrates in tri18 acyltransferase during harzianum A biosynthesis

Trichothecenes are secondary metabolites synthesized by insects, plants, and diverse fungal genera, including Trichoderma species. Fungal acyl/acetyltransferases of the BAHD family contain an evolutionarily conserved HXXXD motif in their central region and serve as crucial catalysts in metabolic and regulatory pathways. In Trichoderma arundinaceum and Trichoderma brevicompactum, two acyl/acetyltransferase enzymes, tri3 and tri18, synthesize trichothecene intermediates trichodermin and harzianum A (HA), respectively. Previous studies demonstrated that tri3 catalyzes acetylation of trichodermol at the C-4 hydroxyl group to produce trichodermin, while tri18 subsequently modifies this acetyl group with an octa-2,4,6-trienedioyl moiety to form harzianum A. However, the catalytic mechanism of tri18 in HA synthesis remains poorly characterized. This study employed computational approaches, including protein modeling and refinement, comparative sequence analysis, molecular docking, molecular dynamics simulations (MDS), and MMPBSA free energy calculations to elucidate tri18 function and substrate specificity in HA biosynthesis. Comparative sequence analysis identified the His223-Gly228 motif responsible for initial substrate binding, consistent with other fungal acyltransferases. Binary and ternary complex analyses revealed differential tri18 behavior with candidate substrates. Molecular docking, MDS, and MMPBSA results demonstrated substrate-specific binding affinity, complex stability, and catalytic mechanisms, providing molecular insights into tri18-mediated acylation. This research elucidates the final step of HA biosynthesis and establishes a foundation for exploiting this pathway in sustainable agriculture, particularly for developing biocontrol agents targeting plant pathogens.