# Early occurrence of caffeoylshikimic acid esterase (CSE) activity in the hornwort Anthoceros agrestis

## TL;DR

A new enzyme in a hornwort plant shows esterase and lipase activity, suggesting an evolutionary step in enzyme specialization.

## Contribution

First report of an active CSE with dual esterase and lipase activity in a non-seed plant.

## Key findings

- AaCSE1 from Anthoceros agrestis has esterase activity with caffeoyl esters and lipase activity with 4-nitrophenyl butyrate.
- AaCSE1 has lower catalytic efficiency compared to Arabidopsis thaliana CSE.
- MeMAGL3 from Mesotaenium endlicherianum shows only lipase activity.

## Abstract

The hornwort Anthoceros agrestis harbors a caffeoylshikimate esterase (CSE) displaying esterase activities with various caffeoyl- and 4-coumaroyl esters as well as lipase activity with the surrogate substrate 4-nitrophenyl butyrate.

Caffeoylshikimic acid esterase (CSE) is an enzyme of the monoacylglycerol lipase (MAGL) family shown to be involved in monolignol biosynthesis and thus has importance for lignification. To date, active CSEs have only been found in seed plants. A protein (AaCSE1) from the hornwort Anthoceros agrestis with 51.5% identity to the respective CSE sequence from Arabidopsis thaliana (AtCSE, At1g52760) displayed esterase activity with caffeoyl-5-O-shikimic acid as well as its 3-O- and 4-O-regioisomers. Chlorogenic acid (caffeoyl-5-O-quinic acid) as well as 4-coumaroyl esters were accepted with a lower affinity and catalytic efficiency. Slight activity could also be demonstrated for cleavage of the amide bond in N-(caffeoyl)-5-hydroxyanthranilic acid. Although AaCSE displays CSE activity and has high affinity for its substrate, this enzyme has a lower catalytic efficiency (~ 21-fold lower) compared to the CSE from Arabidopsis thaliana. Assays with the surrogate lipase substrate 4-nitrophenyl butyrate showed lipase activity. Thus, AaCSE1 could serve a dual function as esterase and lipase. Three putative CSE sequences from Mesotaenium endlicherianum, a model organism from the Zygnematophyceae, were also amplified and heterologously expressed. Only MeMAGL3 was active as a lipase. Our study showed for the first time an active CSE from a non-seed plant with dual activity as esterase/amidase as well as lipase, which could indicate a transitional state towards the evolution of more specialized CSEs predominantly involved in monolignol formation.

The online version contains supplementary material available at 10.1007/s00425-025-04915-7.