Molekularbiologische und biochemische Untersuchungen von Hydroxycinnamoyltransferasen aus Coleus blumei und Glechoma hederacea

Hydroxycinnamic acid esters are prominent natural products in Lamiaceae. Besides hydroxycinnamoylshikimate and -quinate, which are rather ubiquitous in the plant kingdom, rosmarinic acid is found predominantly in Boraginaceae and in the subfamily Nepetoideae of the Lamiaceae. The biosynthetic pathways towards monolignols, chlorogenic acid and rosmarinic acid are extensively investigated. The hydroxycinnamoyltransferases (HCTs), involved in the biosynthesis of these esters, belong to the superfamily of BAHD acyltransferases and accept hydroxycinnamoyl-CoAs as hydroxycinnamoyl donors. They transfer hydroxycinnamic acids from CoA to different acceptor substrates (HST: shikimate, HQT: quinate, RAS hydroxyphenyllactates) and show generally an explicit substrate specificity. The first cDNA encoding a hydroxycinnamoyl-CoA: hydroxyphenyllactate hydroxycinnamoyltransferase (CbRAS) was cloned from Coleus blumei. RAS enzyme activity is essential for the formation of rosmarinic acid. In this work the cDNA encoding a hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (CbHST) could be isolated from the same plant species. The proteins were heterologously expressed in E. coli and the substrate acceptance of both enzymes were tested. Interestingly, besides ester formation also amide formation can be catalyzed. CbHST transfers CoA-activated cinnamic acids (p-coumaroyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, feruloyl-CoA, sinapoyl-CoA) to shikimate but not to quinate or acceptor substrates utilized by CbRAS. Also 3-hydroxyanthranilate, 2,3-dihydroxybenzoate, 3-hydroxybenzoate and 3-aminobenzoate yielded products. CbRAS transfers the same CoA-activated cinnamic acids (except sinapoyl-CoA) to D-(hydroxy-)phenyllactic acids (D-4-hydroxyphenyllactate, D-3,4-dihydroxyphenyllactate, D-phenyllactate), but not to quinate or shikimate. In addition, the D-amino acids D-phenylalanine, D-tyrosine and D-DOPA are accepted as acceptor substrates by CbRAS. The Km-values for a specific substrate were dependent on the second substrate used in the assay. This is an indication of the plasticity of the active sites (induced fit). The coexistence of both enzymes in one species, the high substrate specificity and the kinetic parameters indicate, that CbHST is a specific enzyme of monolignol biosynthesis and CbRAS a specific enzyme of rosmarinic acid biosynthesis. p-coumaroyl-CoA is the preferred substrate. To identify important amino acid residues, CbRAS mutants H152A, D156A, D377A, R285A, W380L and L136P were generated. CbRAS mutants showed a residual activity < 1 % in comparison to the wild type RAS. An involvement of H152 in catalysis is anticipated. To perform crystallization experiments, two different purification procedures were established, which allow the purification of CbRAS to apparent homogeneity. Glechoma hederacea accumulates rosmarinic acid and chlorogenic acid in parallel. Therefore Glechoma is suitable for the detailed investigation of the three biosynthetic pathways. By sequence alignment of known RAS, HST and HQT degenerated primers were designed. The 5´- and 3´-ends were amplified with help of RACE-PCR and enabled the deduction of 6 full-length sequences. All translated protein sequences exhibit the conserved sequence motivs HxxxD and DFGWG. Three enzymes could be expressed functionally in E. coli: GhRAS-l is a hydroxycinamoyl-CoA: hydroxyphenyllactate hydroxycinnamoyltransferase and transfers p-coumaric acid and caffeic acid from CoA to p-hydroxyphenyllactate (pHPL) and 3,4-dihydroxyphenyllactate (DHPL), but not to shikimate or quinate. GhHST-k und GhHST-l transfer both CoA-activated acids to shikimate, but not to pHPL, DHPL or quinate. GhRAS-k has an identity of 91 % to GhRAS-l, but showed no enzyme activity. A pseudogen is supposed. On the basis of the amino acid sequence, the putative GhHQT-k and GhHQT-l have the highest similarity to a HQT from Cynara cardunculus (50 % and 45 %). No enzyme activity could be detected. Possibly GhHQT-k and GhHQT-l are no HQTs, but HCTs with unknown substrate specificity so far. A phylogenetic tree of the isolated RAS-, HST- and putative HQT- sequences from Coleus and Glechoma together with other biochemical characterized BAHD-acyltransferases showes that the HCTs are closely related. The Phylogenie indicates, that RAS, HST and HQT evolved possibly by duplication and diversification.