Characterization of a dihydrocaffeic acid-coenzyme A ligase and three dihydrocaffeoyl acyltransferases catalyzing formation of kukoamine A in Lycium barbarum

Objective Lycii Cortex (Digupi in Chinese) (root bark of Lycium barbarum ), a widely used traditional Chinese medicine, accumulates bioactive phenolamides named kukoamines. The enzymes catalyzing kukoamine formation in L. barbarum remain unknown. This study aims to characterize the key coenzyme A (CoA) ligase and BAHD acyltransferase (a plant-specific acyltransferase family) that catalyze formation of kukoamine A. Methods Candidate genes were identified using BLAST and phylogenetic analysis. Catalytic functions and substrate specificities were characterized through recombinant protein expression, in vitro enzymatic assays, and chromatographic and mass spectrometric analyses. Molecular modeling and site-directed mutagenesis were performed to identify key residues involved in catalysis. Results Three BAHD acyltransferases (BAHD-ATs), L. barbarum acyltransferase 1/6/7 (LbAT1/6/7), along with a dihydrocaffeic acid - coenzyme A ligase, L. barbarum CoA ligase 1 (Lb4CL1), were identified. LbAT1/6/7 represent the first functionally characterized dihydrocaffeoyl-CoA: spermine acyltransferases that catalyze kukoamine A biosynthesis. Phylogenetic analysis revealed that they showed distinct phylogenetic divergence from most characterized polyamine BAHD-ATs. Further mining of the One Thousand Plant Transcriptomes Project (1KP) database identified a lineage-specific expansion of these polyamine BAHD-AT genes within the Solanaceae family. LbAT6 exhibited broad substrate promiscuity, accepting multiple polyamine substrates and diverse hydroxycinnamoyl-CoA derivatives as acyl donors. Structural modeling and site-directed mutagenesis identified critical residues governing the catalytic activities of LbAT6 and Lb4CL1. Conclusion This study identified Lb4CL1 and LbAT1/6/7 as the key enzymes catalyzing the formation of kukoamine A, and laid a foundation for the production of kukoamine A as well as its derivatives.