Untersuchung zur Biosynthese aromatischer Sekundärmetabolite in Zellstrukturen von Sorbus aucuparia L. und Centaurium erythraea RAFN. (English shop)

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Benzoic acid and benzoic acid derivatives can be formed in plants via a wide variety of biosynthetic pathways. Highly diverse mechanisms have been described for different plants. The elucidation of the enzymes involved, together with the associated description of the reaction sequence of the individual biosynthetic pathways, has so far only been achieved to a limited extent. Cell cultures of Centaurium erythraea provide an interesting model system for investigating the biosynthesis of 3-hydroxybenzoic acid via intermediates of the shikimate pathway.

In the present work, the ATP-dependent biosynthesis of 3-hydroxybenzoic acid from shikimic acid was demonstrated for the first time in biochemical studies. The enzymatic product was detected exclusively in elicitor-treated cell cultures of C. erythraea, which, in addition to the constitutively formed 1-hydroxy-3,5,6,7,8-pentamethoxyxanthone, also accumulate 1-hydroxy-3,5,6,7-tetramethoxyxanthone.

Investigations into the biosynthesis of benzoic acid were also carried out, in which p-coumarate:CoA ligases (4CL) in cell cultures of Sorbus aucuparia were studied. In higher plants, 4CL is encoded by gene families. A specialisation of the individual isoforms is suggested by the differing substrate specificities, expression patterns and structural organisation of their genes. In some plants, 4CL isoforms can thus be assigned to lignin biosynthesis or to other phenylpropanoid pathways. Despite their differing substrate specificities, the activity spectrum of all known 4CLs is restricted to cinnamic acid derivatives.

Here, Sa4CL2, the second p-coumarate:CoA ligase from S. aucuparia, was identified. The ORF comprises 1644 bp and encodes 547 amino acids. Sa4CL2 has a molecular mass of approximately 62 kDa. The highest catalytic efficiency (kcat/KM) is shown towards ferulic acid and caffeic acid, followed by p-coumaric acid and cinnamic acid. Sinapic acid and benzoic acid are not accepted. The pH optimum of the reaction is pH 7.5 and the temperature optimum 35–40 °C. The reaction is dependent on divalent cations, with the highest activities being achieved with manganese and magnesium.

For the three Sa4CL isoforms known to date, differential expression in different tissues was demonstrated here. Sa4CL2 and Sa4CL3 additionally exhibit differing substrate specificities. Together with significant differences in the primary structure of the proteins, Sa4CL1 and Sa4CL2 can be assigned to class I of the 4CLs (lignin biosynthesis) and Sa4CL3 to class II (flavonoid biosynthesis).

A massive increase in 4CL expression does not occur in elicitor-treated cell cultures of S. aucuparia, in contrast to the strong induction of biphenyl synthase. Sa4CLs therefore appear to be involved neither in the biosynthesis nor in the activation of benzoic acid. A classical recruitment of the 4CLs for lignin biosynthesis and other phenylpropanoid metabolic pathways is suggested.

Following pathogen attack, S. aucuparia forms phytoalexins of the biphenyl type. The most abundant biphenyl in quantitative terms is aucuparin. In this work, a synthetic scheme was developed which, in addition to aucuparin, also gives access to the isomeric dihydroxy-monomethoxybiphenyls and the isomeric monohydroxy-dimethoxybiphenyl. Starting from a Suzuki coupling, possible intermediates of aucuparin biosynthesis can thus also be readily prepared. These may be valuable as substrates for the investigation of O-methyltransferase reactions.