Orthology‐based analysis helps map evolutionary diversification and predict substrate class use of BAHD acyltransferases

Abstract

SUMMARY Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast‐evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG‐based analysis enabled identification of signature motifs in anthocyanin‐acylating BAHDs, whose importance was validated via molecular dynamic simulations, site‐directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration‐enabled approach for improved functional annotation of plant enzyme families.

Comprehensive characterization of the complex BAHD acyltransferase family from 218 land plants species: phylogenomic analysis and identification of specificity determinant positions

“…Both training and complete BAHDome sets were used to understand different phylogenetic and functional traits of the BAHD acyltransferase superfamily. In general, the topology of the phylogenetic reconstruction generated (both with training and complete BAHDome sets) agree with others published elsewhere [21], [24]. These studies were dedicated to few species and, since they were mostly based on characterized BAHD enzymes, the complete BAHDome space was not extensively covered.…”

“…These sites implicate an evolutionary advantage towards promiscuity and eventually functional diversification. In addition, a recent study identified residues W36 and C320 in Dm3MaT3 as key for the enzyme's activity [24]. This enzyme corresponds to A4PHY4 from Chrysanthemum morifolium in our dataset, which belongs to G2, and we indeed identified both these positions as SDPs (corresponding to SDPs 31 and 302) in different analyses.…”

“…In G4-3, on the other hand, two SP entries are characterized: O64470 from A. thaliana , with a role in the synthesis of conjugates of hydroxycynnamoyl and spermidine in tapetum cells [34], and B6D7P2 from Trifolium pratense , involved in the biosynthesis of phaselic acid (using coumaroyl-CoA and malate as donor and acceptor, respectively) [35]. It has been recently suggested that the OH-acylation of aliphatic and aromatic alcohols may represent the ancestral set of acceptors for land plants BAHD family, as it most likely existed before land plants emergence [24]. Our results agree with this observation, and in addition offer the notion that CoA-activated hydroxycinnamic acids constitute the ancestral donors.…”

Improved production of the antidiabetic metabolite montbretin A in Nicotiana benthamiana: discovery, characterization, and use of Crocosmia shikimate shunt genes

“…To identify montbretia enzymes involved in the biosynthesis of caffeic acid and caffeoyl-CoA, we used BLASTP searches against a translated transcriptome database that covers a time course of corm development, previously described in Irmisch et al (2020). Known plant HCT sequences typically belong to Clade 5 of the BAHD acyltransferase (AT) family (Kruse et al, 2022(Kruse et al, , 2023Moghe et al, 2023). A search of the montbretia transcriptome using A. thaliana HCT (NP_199704.1) revealed three candidate genes, for which we cloned the coding sequences (CDS) from young corms.…”

“…Ni-affinity purified proteins (Figure S9) were used for enzyme assays, and products were identified based on accurate mass and authentic standards. CcAT8 was assayed with the acyl acceptor shikimate or quinate, and one of the acyl donors caffeoyl-, coumaroyl-, or feruloyl-CoA, which are substrates typically used by HCTs from other plant species (Kruse et al, 2022(Kruse et al, , 2023Moghe et al, 2023). With shikimate as the acyl acceptor, all three acyl-CoA donors were converted into the respective shikimate esters, caffeoylshikimate (m/z 335.0893), coumaroylshikimate (m/z 319.0807) and feruloylshikimate (m/z 349.0932) (Figure 4).…”