# Identification and characterization of BAHD hydroxycinnamoyltransferases in the fern Neoblechnum brasiliense

## TL;DR

This study identifies and characterizes nine BAHD acyltransferases in the fern Neoblechnum brasiliense, revealing their roles in transferring hydroxycinnamoyl units onto various substrates.

## Contribution

The paper reports the first detailed characterization of BAHD hydroxycinnamoyltransferases in the fern Neoblechnum brasiliense.

## Key findings

- Nine BAHD acyltransferases were identified and heterologously expressed in N. brasiliense.
- NbHCT1 and 2 showed high catalytic efficiency with shikimic acid as the acceptor substrate.
- Phylogenetic analysis grouped several enzymes into clade V, known for shikimate and quinate hydroxycinnamoyltransferases.

## Abstract

Neoblechnum brasiliense, a leptosporangiate fern of the family Blechnaceae, is known to have a rich phenolic metabolism. Rosmarinic acid and blechnic acid B have been detected beside chlorogenic acid. As BAHD acyltransferases often are responsible for the formation of this kind of esters, for example, rosmarinic acid synthases or 4‐coumaroyl‐CoA:shikimic or quinic acid hydroxycinnamoyltransferases, nine coding sequences for BAHD acyltransferases were amplified from N. brasiliense and heterologously expressed. All enzymes proved to be active with p‐coumaroyl‐CoA as acyl donor as well as other hydroxycinnamoyl‐CoA derivatives. Four enzymes (NbHCT4, 6, 7, 9) accepted various alcohols and amines but only to a very low extent. High catalytic efficiency was observed for NbHCT1 and 2 that preferred shikimic acid as acceptor substrate, while NbHCT3 was most active with quinic acid and is part of a lineage‐specific duplication subclade of clade V with sequences of Cyatheales and Polypodiales. Tryptamine amides were formed by NbHCT5. NbHCT8 not only preferred 3‐hydroxyanthranilate but also transformed shikimic and quinic acids. NbHCTs 1, 3, and 8 were fully kinetically characterized. In a phylogenetic tree comprising members of all known BAHD clades, NbHCT1, 2, 3, and 8 were grouped into clade V harboring most known shikimate and quinate hydroxycinnamoyltransferases, while NbHCT6 and 7 were in or near clade VI. NbHCT4 was placed in clade IV, NbHCT5 most probably in clade IV and NbHCT9 in clade I. Since no hydroxycinnamoyltransferase forming rosmarinic acid could be identified, further hydroxycinnamoyltransferases must be expected in N. brasiliense.

Nine BAHD acyltransferases have been identified and characterized in the fern Neoblechnum brasiliense transferring hydroxycinnamoyl units onto various alcohol and amine acceptor substrates. Kinetic characterization showed that shikimic and quinic acids for ester formation as well as 3‐hydroxyanthranilic acid and tryptamine for amide formation are the main substrates.