Mechanistic basis for the emergence of EPS1 as a catalyst in plant salicylic acid biosynthesis

Unique to plants in the Brassicaceae family, the production of the plant defense hormone salicylic acid (SA) from isochorismate is accelerated by an evolutionarily young isochorismoyl-glutamate pyruvoyl-glutamate lyase, EPS1, which belongs to the BAHD acyltransferase protein family. Here, we report the crystal structures of apo and substrate-analog-bound EPS1 from Arabidopsis thaliana. Assisted by microsecond molecular dynamics simulations, we uncover a unique pericyclic rearrangement lyase mechanism facilitated by the active site of EPS1. We reconstitute the isochorismate-derived pathway of SA biosynthesis in Saccharomyces cerevisiae, which serves as an in vivo platform that helps identify active-site residues critical for EPS1 activity. This study describes the birth of a new catalyst in plant phytohormone biosynthesis by reconfiguring the ancestral active site of a progenitor enzyme to catalyze alternative reaction.One sentence summaryBy reconfiguring the active site of a progenitor acyltransferase-fold, EPS1 acquired the unique, evolutionarily new lyase activity that accelerates phytohormone salicylic acid production in Brassicaceae plants.

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall

The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total pCA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that pCA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the pCA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in pCA content on hemicelluloses and generated a C4 plant that is promising for valorizing pCA production in biorefineries.

Arabidopsis ECERIFERUM2-LIKEs Are Mediators of Condensing Enzyme Function

Condensing enzymes catalyze the committed reaction of fatty acid elongation and determine the chain length of fatty acids accepted and produced by the elongation complex. While necessary for the elongation of very-long-chain fatty acids (VLCFAs), identified plant condensing enzymes cannot efficiently produce VLCFAs longer than 28 carbons, which are precursors for the most abundant cuticular waxes of most plant species that have been surveyed. The eceriferum2 (cer2) mutant of Arabidopsis thaliana has a severe wax-deficient phenotype and specifically lacks waxes longer than 28 carbons, but the CER2 protein does not share sequence similarity with condensing enzymes. Instead, CER2 is homologous to BAHD acyltransferases. Heterologous expression in yeast previously demonstrated that CER2, and a small clade of BAHD acyltransferases with high sequence identity to CER2, can extend the chain-length specificity of the condensing enzyme CER6. This biochemical function is distinct from that of the broader BAHD acyltransferase family. The product specificity and physiological functions of individual CER2-LIKE proteins are unique. Here, we demonstrate that CER2 physically interacts with the fatty acid elongase. We cloned chimeric CER2-LIKE proteins and expressed these in yeast cells to identify the features that define the substrate specificities of CER2-LIKEs. We generated homology-based structural models to compare CER2-LIKEs and BAHD acyltransferases. In addition, based on the current phylogenetic analysis of the CER2-LIKE clade, we describe two further Arabidopsis CER2-LIKE genes, CER2-LIKE3 and CER2-LIKE4. We used yeast expression and mutant analysis to characterize these genes. Collectively, these results expand our knowledge of the functions of CER2-LIKEs, the BAHD acyltransferase family and cuticular wax metabolism.

Ancestral class-promiscuity as a driver of functional diversity in the BAHD acyltransferase family in plants

ABSTRACTGene duplication-divergence and enzyme promiscuity drive metabolic diversification in plants, but how they contribute to functional innovation in enzyme families is not clearly understood. In this study, we addressed this question using the large BAHD acyltransferase family as a model. This fast-evolving family, which uses diverse substrates, expanded drastically during land plant evolution. In vitro characterization of 11 BAHDs against a substrate panel and phylogenetic analyses revealed that the ancestral enzymes prior to origin of land plants were likely capable of promiscuously utilizing most of the substrate classes used by current, largely specialized enzymes. Motif enrichment analysis in anthocyanin/flavonoid-acylating BAHDs helped identify two motifs that potentially contributed to specialization of the ancestral anthocyanin-acylation capability. Molecular dynamic simulations and enzyme kinetics further resolved the potential roles of these motifs in the path towards specialization. Our results illuminate how promiscuity in robust and evolvable enzymes contributes to functional diversity in enzyme families.

Global dissection of the BAHD acyltransferase gene family in soybean: Expression profiling, metabolic functions, and evolution

Background: Members of the BAHD acyltransferase (ACT) family play important roles in plant defence against biotic and abiotic stresses. A better understanding of the functions that specific members of this family play in stress defence can lead to better breeding strategies for stress tolerance. Previous genome-wide studies explored other acyltransferase families, but so far not a single study has been published on genome-wide or positive selection analyses of the BAHD genes in Glycine max . Results: A total of 103 genes of the BAHD family (GmACT genes) were identified from the soybean genome, which could be grouped into four phylogenetic clades (I-IV). Clade III was further divided into two sub-clades (IIIA and IIIB). In each clade, the gene structure and functional motifs were relatively well conserved. These ACT genes were unequally distributed on all 20 chromosomes, and 16 paralogous pairs were found within the family. Positive selection analysis revealed amino acids under strong positive selection, which suggests that the evolution of this gene family modulated soybean domestication. Some GmACT genes showed expression specific under specific conditions, while others showed constitutive expression in all soybean tissues or conditions analysed. Most of the expression of ACT genes in soybean was repressed with Al 3+ and fungal elicitor exposure, except for GmACT84 , which expression increased in these conditions 2- and 3-fold, respectively. The promoter of GmACT84 contains the maximum number of stress-responsive elements among all GmACT genes and it is especially enriched in MYB-related elements. Conclusions: This study provides a genome-wide analysis of the BAHD gene family and assessed their expression profiles in soybean. We found evidence of a strong positive selection of GmACT genes. Our findings will help efforts of functional characterisation of ACT genes in soybean in order to discover their involvement in growth, development, and defence mechanisms.

Global dissection of the BAHD acyltransferase gene family in soybean: Expression profiling, metabolic functions, and evolution

Background: Members of the BAHD acyltransferase (ACT) family play important roles in plant defence against biotic and abiotic stresses. Previous genome-wide studies explored different acyltransferase gene families, but not a single study was found so far on the overall genome-wide or positive selection analyses of the BAHD family genes in Glycine max . A better understanding of the functions that specific members of this family play in stress defence can lead to better breeding strategies for stress tolerance. Results: A total of 103 genes of the BAHD family (GmACT genes) were mined from the soybean genome, which could be grouped into four phylogenetic clades (I- IV). Clade III was further divided into two sub-clades (IIIA and IIIB). In each clade, the constituent part of the gene structures and motifs were relatively conserved. These 103 genes were distributed unequally on all 20 chromosomes, and 16 paralogous pairs were found within the family. Positive selection analysis revealed important amino acids under strong positive selection, which suggests that the evolution of this gene family modulated soybean domestication. Most of the expression of ACT genes in soybean was repressed with Al 3+ and fungal elicitor exposure, except for GmACT84 , which expression increased in these conditions 2- and 3-fold, respectively. The promoter region of GmACT84 contains the maximum number of stress-responsive elements among all GmACT genes and is especially enriched in MYB-related elements. Some GmACT genes showed expression specific under specific conditions, while others showed constitutive expression in all soybean tissues or conditions analysed. Conclusions: This study provided a genome-wide analysis of the BAHD gene family and assessed their expression profiles. We found evidence of a strong positive selection of GmACT genes. Our findings will help efforts of functional characterisation of ACT genes in soybean in order to discover their involvement in growth, development, and defence mechanisms.