Hydroxycinnamoyl-coenzyme A: tetrahydroxyhexanedioate hydroxycinnamoyl transferase (HHHT) from Phaseolus vulgaris: Phylogeny, expression pattern, kinetic parameters, and active site analysis

Authors: FANELLI, AMANDA - Oak Ridge Institute For Science And Education (ORISE); Arther, Christina; Sullivan, Michael

Submitted to: Life Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2025
Publication Date: 2/20/2025
Citation: Fanelli, A., Arther, C.M., Sullivan, M.L. 2025. Hydroxycinnamoyl-coenzyme A: tetrahydroxyhexanedioate hydroxycinnamoyl transferase (HHHT) from Phaseolus vulgaris: Phylogeny, expression pattern, kinetic parameters, and active site analysis. Life Sciences. https://doi.org/10.7717/peerj.19037.
DOI: https://doi.org/10.7717/peerj.19037

Interpretive Summary: BAHD acyl-Coenzyme A (CoA) transfereases comprise a large family of enzymes in plants which transfer an acyl group from a CoA thioester donor to hydroxyl or amine groups to form esters or amides, respectively. The specialized metabolites made by these enzymes are involved in functions such as structure (e.g. lignin formation) and mitigating biotic and abiotic stress and many have bioactive properties and could be useful as nutriceuticals. The diversity of these enzymes makes it difficult to predict substrate specificity and function based on gene sequence alone and relatively few have been characterized biochemically. Here we characterized a BAHD transferase from bean with respect to its gene expression, kinetic parameters (substrate specificity), and predicted three-dimensional (3-D) structure and active site interactions with acceptor substrates. Our findings provide a foundation for better understanding how 3-D structure of BAHD acyl-CoA transferases relates to their substrate specificity, which could ultimately lead to better prediction of their function and ability to rationally design and engineer BAHD transferases to make useful and novel products.

Technical Abstract: BAHD acyl-coenzyme A (CoA) transferases comprise a large family of enzymes in plants which transfer an acyl group from a CoA thioester to hydroxyl or amine groups to form esters or amides, respectively. Clade Vb of this family primarily utilizes hydroxycinnamoyl-CoA as the acyl donor. These enzymes are involved in biosynthesis of diverse specialized metabolites with functions such as structure (e.g., lignin formation) and biotic/abiotic stress mitigation. The diversity of these enzymes has arisen from both divergent and convergent evolution, making it difficult to predict substrate specificity or enzyme function based on homology, and relatively few BAHD transferases have been characterized biochemically with respect to substrate specificity. We previously identified a hydroxycinnamoyl-CoA: tetrahydroxyhexanedioate hydroxycinnamoyl transferase (HHHT) from common bean capable of transferring hydroxycinnamic acids to mucic or saccharic acid to form the corresponding esters. Here, to better understand the structure/function relationships of this enzyme, we have further characterized it with respect to expression pattern, kinetic parameters, and predicted three-dimensional (3-D)structure and active site interactions with acceptor substrates. The hhht gene was expressed predominantly in leaves and to a lesser extent flowers and shoots. KM values did not vary greatly among donor or among acceptor substrates generally less than two-fold), while kcat values were consistently higher for saccharic acid as substrate compared to mucic acid, leading to higher catalytic efficiency (as kcat/KM)for saccharic acid. Both acceptors had similar binding poses when docked into the active site, and the proximity of multiple hydroxyl groups to the catalytic His 150, especially for saccharic acid, might provide some insights into regiospecificity. These findings provide a foundation for better understanding how the 3-D structure of BAHD transferases relates to their substrate specificity, as we explore the chemistry of the active site and interactions with ligands. This could ultimately lead to better prediction of their function and ability to rationally design BAHD transferases to make useful and novel products.