Identification and characterization of three acyltransferases involved in the hydroxycinnamic acid amide synthesis of goji berry.

Hydroxycinnamic acid amides (HCAAs) featuring spermine or spermidine represent a major class of specialized metabolites in goji berries (Lycium spp.) and exhibit diverse pharmacological activities. Acylation is essential for HCAAs biosynthesis, yet the underlying enzymatic mechanisms in goji berries remain unclear. In this study, three acyltransferase genes (LaSHT1, LaSHT2 and LcSHT) were cloned from goji berries. In vitro assays revealed that all three enzymes catalyze the formation of double N-acylated HCAAs exhibiting distinct substrate preferences. Further biochemical characterization of the recombinant proteins determined their optimal catalytic conditions and kinetic parameters, thereby defining their substrate specificities toward spermine and spermidine. Gene expression analysis revealed that LaSHT1, LaSHT2, and LcSHT are differentially expressed across tissues, suggesting their distinct physiological roles in HCAAs accumulation in goji. To elucidate the structural basis of substrate recognition, we analyzed the substrate binding of LaSHT2 and LcSHT using homology modeling, molecular docking, and molecular dynamics simulations, which revealed the distances, stabilities, and conformations of substrates within these active pockets. Site-directed mutagenesis identified residues that strongly influence catalytic activity, providing preliminary insights into the SHT catalytic mechanism for recognizing diverse polyamines. Together, these results provide insight into the enzymatic basis of HCAA biosynthesis in goji berry.