Structural and Biochemical Insights Into Two BAHD Acyltransferases (AtSHT and AtSDT) Involved in Phenolamide Biosynthesis

Abstract

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 AtSHT and AtSDT, two representative BAHD-family members that catalyze the multisite acylation of spermidine but show different product profiles. By determining the structures of AtSHT and AtSDT and using structure-based mutagenesis, we identified the residues important for substrate recognition in AtSHT and AtSDT and hypothesized that the acyl acceptor spermidine might adopt a free-rotating conformation in AtSHT, which can undergo mono-, di-, or tri-acylation; while the spermidine molecule in AtSDT 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 Arabidopsis 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.

Characterization of evolutionarily distinct rice BAHD‐Acyltransferases provides insight into their plausible role in rice susceptibility to Rhizoctonia solani

“…The plant BAHD family enzymes have complex substrates (a variety of acyl‐donors and acceptors) and different products. It is very difficult to predict the substrate specificity of uncharacterized BAHD family enzymes because (a) of the minimal sequence similarity among the members of BAHD gene family and (b) the multisite acylation pathways of BAHD‐family acyltransferases are poorly understood (Bontpart et al., 2015; Chiang et al., 2018; Wang et al., 2020). The first BAHD gene to be functionally identified was glossy2 ( Zea mays L.) /CER2 [ Arabidopsis thaliana (L.) Heynh.…”

Biosynthesis of strychnine

“…14 ). Homology models of Snv AT and Sp AT 29 (Supplementary Fig. 15 ) were used to identify one amino acid ( Snv AT(R424F) and Sp AT(F421R)) that controls the selectivity between acetyl and malonyl transferase activity (Supplementary Figs.…”

Functional characterization of a small gene family coding for putrescine hydroxycinnamoyltransferases in tomato

“…The expression of these genes was described to be dramatically induced in response to herbivory and to contribute to an increase of the accumulation of caffeoylputrescine in the damaged tissues (Roumani et al 2022). Such genes coding for PHTs were already described in N. attenuata (Onkokesung et al 2012), in Arabidopsis (Wang et al 2021) and in rice where this activity was also supported by a small gene family displaying complementary expression profile (Dong et al 2015;Tanabe et al 2016). The aim of this study was to characterize the determinants of putrescine-derived phenolamide accumulation in tomato plants.…”