Comparativemolecular and biochemical characterization of acyltransferases, hydrolases and ligases from anthoceros agrestis and mesotaenium endlicherianum
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The terrestrialization of plants marks one of the most impactful evolutionary transitions in the history of life on this planet. The ability of these early organisms to survive in harsh environments might have been partly linked to the evolution of specialized phenolic metabolites, which can provide limited protection against stressors such as UV radiation, drought and pathogens. Therefore, gaining a deeper understanding of the phenylpropanoid pathway in extant aquatic and terrestrial plants may provide useful insights into the adaptations required for the colonization of dry land. The central compound studied in this work was rosmarinic acid, as it likely contributes to plant stress tolerance against the biotic and abiotic challenges described above. This study focused on the identification, comparison and characterization of enzymes related to the phenylpropanoid pathway in the hornwort Anthoceros agrestis and the alga Mesotaenium endlicherianum. Four novel hydroxycinnamoyltransferase (HCT) sequences were elucidated in A. agrestis (HCT8, HCT9, HCTA and HCTF), two of which were characterized in more detail. AaHCT8 catalyzes the formation of 4-coumaroyl-5-O-shikimic acid, 4-coumaroyl-5-O-quinic acid, 4-coumaroyl-3,4-dihydroxybenzoic acid and 4-coumaroylmalic acid. It represents the first active embryophyte HCT (and BAHD) assigned to clade 0, a lineage previously considered to exclusively be comprised of algal sequences. AaHCTA also facilitated the formation of 4-coumaroyl-5-O-shikimic acid and 4-coumaroyl-3,4-dihydroxybenzoic acid. In addition, it produced all three regioisomers of caffeoylshikimic acid (3-O, 4-O and 5-O) and showed an unusual basic pH optimum at pH 9.0. Three A. agrestis serine carboxypeptidase-like acyltransferases (SCPLs) were expressed heterologously, but none of them were able to catalyze acyltransferase reactions associated with the formation of rosmarinic acid when supplied with hydroxycinnamic glucose esters as donor substrates. This was repeated in both prokaryotic and eukaryotic expression systems, yielding similar results. Phylogenetic analysis revealed AaSCPL1 and AaSCPL2 to cluster within clade IB, in which functionality has yet to be successfully identified. A. agrestis is the first bryophyte in which an active caffeoylshikimate esterase (CSE) has been successfully characterized. AaCSE1 was shown to catalyze the hydrolysis of caffeoylshikimic acid and caffeoylquinic acid (chlorogenic acid), as well as their regioisomers (3-O, 4-O and 5-O). Additionally, it was able to cleave 4-coumaroyl-5-O-shikimic acid and 4-coumaroyl-5-O- quinic acid in trace amounts. A direct comparison of catalytic efficiencies revealed a pronounced preference for caffeic acid esters, with caffeoyl-3-O-shikimic acid being the most efficiently hydrolyzed regioisomer. 4-Nitrophenyl butyrate (4-NPB) was used as a surrogate substrate to test for lipase (MAGL) activity. This study provides compelling experimental evidence suggesting that AaCSE1 exhibits lipase activity in addition to esterase activity, which had not previously been observed in CSEs. Structural analysis was performed to identify potentially significant amino acid residues that facilitate the substrate promiscuity. Three potential CSE/MAGL sequences of M. endlicherianum were elucidated and lacked detectable esterase activity; however, MeMAGL3 hydrolyzed 4-NPB and was therefore classified as a lipase. Finally, no convincing phenylalanine ammonia-lyase (PAL) sequence or active 4-coumarate CoA-ligase (4CL) was identified in M. endlicherianum. A putative HAL sequence was elucidated within an early version of the M. endlicherianum transcriptome and was subsequently expressed and characterized. PCR amplification yielded no result, and bioinformatic analysis showed a high identity with an unknown Sediminibacterium sequence. This suggests that the gene is unlikely to have originated from M. endlicherianum. Additionally, two putative 4CLs were identified and expressed. Me4CL1 was adapted in multiple iterations, without successfully establishing activity. In contrast, Me4CL2 was enzymatically active but functioned as an oxalate CoA-ligase (OCL) rather than a 4CL; it was therefore reclassified and labeled as MeOCL2. This marks the first OCL ever characterized in a non-vascular plant.
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