Structural and Biochemical Insights Into Two BAHD Acyltransferases (SHT and SDT) Involved in Phenolamide Biosynthesis.

Phenolamides represent one of the largest classes of plant-specialized secondary metabolites and function in diverse physiological processes, including defense responses and development. The biosynthesis of phenolamides requires the BAHD-family acyltransferases, which transfer acyl-groups from different acyl-donors specifically to amines, the acyl-group acceptors. However, the mechanisms of substrate specificity and multisite-acylation of the BAHD-family acyltransferases remain poorly understood. In this study, we provide a structural and biochemical analysis of SHT and SDT, two representative BAHD-family members that catalyze the multisite acylation of spermidine but show different product profiles. By determining the structures of SHT and SDT and using structure-based mutagenesis, we identified the residues important for substrate recognition in SHT and SDT and hypothesized that the acyl acceptor spermidine might adopt a free-rotating conformation in SHT, which can undergo mono-, di-, or tri-acylation; while the spermidine molecule in SDT might adopt a linear conformation, which only allows mono- or di-acylation to take place. In addition, through sequence similarity network (SSN) and structural modeling analysis, we successfully predicted and verified the functions of two uncharacterized BAHD acyltransferases, OAO95042.1 and NP_190301.2, which use putrescine as the main acyl-acceptor. Our work provides not only an excellent starting point for understanding multisite acylation in BAHD-family enzymes, but also a feasible methodology for predicting possible acyl acceptor specificity of uncharacterized BAHD-family acyltransferases.