Identification of a hydroxycinnamoyl-CoA:tartarate hydroxycinnamoyltransferase from perennial peanut (Arachis glabrata)

Authors: Koch, Lisa; Reinhardt, Laurie; FANELLI, AMANDA - Orise Fellow; Sullivan, Michael

Submitted to: Phytochemical Society of North America Meeting and Newsletter
Publication Type: Abstract Only
Publication Acceptance Date: 3/6/2026
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: An array of hydroxycinnamoyl esters and amides accumulates in tissues of various plant species where they serve roles in protection against biotic and abiotic stress. Caffeic acid derivatives in particular can be oxidized by endogenous polyphenol oxidase (PPO) enzymes. In forage crops, reactive quinones resulting from this oxidation improve protein preservation during storage and improved protein utilization by ruminant animals consuming the forage. We previously identified PPO activity, caffeoyl-tartarate (caftaric acid) PPO substrate as well as other hydroxycinnamoyl-tartarate esters, and a hydroxycinnamoyl-CoA:tartarate transferase (HTT) activity in perennial peanut (Arachis glabrata) leaves. To identify candidates for an HTT gene and assess their expression levels, we used Illumina sequencing and Trinity to generate a de novo transcriptome assembly from unexpanded and mature perennial peanut leaves. Among the assembled mRNAs, we identified eight clade Vb BAHD acyltransferases as potential HTT candidates, as well as one with a high degree of sequence similarity (90%) to a previously characterized red clover hydroxycinnamoyl-CoA:shikimate transferase (HST). We subsequently confirmed HST activity of this enzyme by expression of the corresponding cDNA in Escherichia coli. Among the other eight candidates, one had the highest expression levels in leaf tissues, and when expressed in E. coli, produced a protein with HTT activity (AgHTT) for which we determined kinetic parameters. Although AgHTT shares 51-54% sequence identity with legume hydroxycinnamoyl-CoA transferases utilizing malate, tyrosine, or tetrahydroxyhexanedioate acceptors, it has relatively low sequence identity (35%) with HTT from Echinacia purpurea, underscoring that primary sequence identity is not always predictive of function for this class of enzymes. Considering this, we predicted AgHTT three-dimensional structure using Alphafold and conducted molecular docking of tartaric acid into the enzyme active site, finding structural aspects that can be related to acceptor substrate specificity. Expression of the AgHTT cDNA in alfalfa, which normally does not accumulate hydroxycinnamoyl derivatives in its leaves, did not result in accumulation of hydroxycinnamoyl-tartaric acid esters. However, incubation of p-coumaroyl-CoA and tartaric acid in leaf extracts from these alfalfa plants resulted in formation of p-coumaroyl-tartarate, suggesting alfalfa leaves lack the required tartarate acceptor. Although we sought to use AgHTT to produce caftaric acid in alfalfa for improved protein preservation and utilization in diary forage systems, lack of tartaric acid acceptor in alfalfa would require adding additional pathways to provide it. However, our identification and functional characterization of AgHTT, which is significantly different from Echinacea HTT in terms of primary structure, provides additional evidence of convergent evolution in the BAHD acyltransferase family and insights into structural features dictating substrate specificity. These insights could help allow rational design of BAHD acyltransferases to produce novel and useful specialized metabolites.