Investigations on the origin of the 3,4-dihydroxyphenyllactic acid moiety of rosmarinic acid in Anthoceros agrestis

Plant terrestrialisation is closely linked to the evolution of phenolic secondary metabolites, which conferred a survival advantage in the presence of new environmental factors, e.g. UV-B radiation, drought, pathogens and herbivores. Besides the incorporation of phenolic components into structural e...

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1. Verfasser: Busch, Tobias
Beteiligte: Petersen, Maike (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2022
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Zusammenfassung:Plant terrestrialisation is closely linked to the evolution of phenolic secondary metabolites, which conferred a survival advantage in the presence of new environmental factors, e.g. UV-B radiation, drought, pathogens and herbivores. Besides the incorporation of phenolic components into structural elements (cuticle, spore wall, cell wall), the esters of caffeic acid are of importance (Weng and Chapple 2010). Rosmarinic acid, formally an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, has been shown to have anti-inflammatory, antimicrobial, antitumour and antioxidant effects, among others (Amoah et al. 2016; Hitl et al. 2021). Besides being abundant in Lamiaceae (Nepetoideae) and Boraginaceae, the component is widespread in the plant kingdom, with hornworts, e.g. Anthoceros agrestis, as the most basal representatives (Petersen 2013). Within the scope of this work, the two enzymes tyrosine aminotransferase (TAT) and hydroxyphenylpyruvate reductase (H(P)PR) were identified, heterologously expressed in E. coli and enzymatically characterised. TAT revealed a molecular mass of about 50 kDa, with the active enzyme being expected as homodimer. The highest enzyme activity was measured at a pH value of 7.9-8.4 at 60 °C. The most probable substrate/co-substrate pair was determined to be L-tyrosine/2-oxoglutarate. Depending on the actual substrate concentrations, phenylpyruvate could also be considered. Oxaloacetate and pyruvate, on the other hand, were only poorly accepted. In addition, several aromatic and aliphatic amino acids were accepted, as well as the keto acid prephenate. For the identification of these substrates, a rapid and reliable method via thin-layer chromatography with detection by ninhydrin staining verified through HPLC and derivatisation with o-phthalaldehyde was introduced. HPPR revealed a molecular mass of about 35 kDa and is as well expected as homodimer. The highest enzyme activities were measured at a pH of 7.0-7.5 at a temperature of 44 °C. Contrary to early expectations, hydroxypyruvate was determined as the primary substrate with NADPH as favoured co-substrate and not 4-hydroxyphenylpyruvate. According to the enzyme family, only the D-enantiomer of 4-hydroxyphenyllactate was formed during the reaction. The results correspond to observations on heterologously expressed H(P)PR from Coleus blumei. In addition, the 3-hydroxylated and methoxylated derivative of 4-hydroxyphenylpyruvate as well as phenylpyruvate and pyruvate were accepted. While no further isoforms could be found for TAT in Anthoceros agrestis, HPPR2 as well as the related HPR1, the photorespiratory isoenzyme, could be amplified and heterologously expressed in E. coli. HPPR2 showed comparable activity with hydroxypyruvate and 4-hydroxyphenylpyruvate, while HPR1 did not accept the latter and favoured NADH. The investigated enzymes TAT, HPPR as well as HPPR2 could be involved in the biosynthesis of the 3,4-dihydroxyphenyllactic acid moiety analogous to Coleus blumei. Expression analysis over a 14-day culture period revealed a parallel progression of TAT and HPPR2 transcript abundance and rosmarinic acid content, which was different from previous observations in different suspension cultures of Anthoceros agrestis. A definite statement about the involvement of the enzymes mentioned cannot be made based on these data. However, the identification of the potential core enzymes has laid a solid foundation for future work focusing on in vivo analyses.
DOI:10.17192/z2023.0078