Abstract
Main conclusionAnthoceros agrestis hydroxycinnamoyltransferase accepts shikimic and 3-hydroxyanthranilic acids while hydroxycinnamoylester/amide 3-hydroxylase (CYP98A147) preferred p-coumaroyl-(3-hydroxy)anthranilic acid compared to the shikimic acid derivative. Alternative pathways towards rosmarinic acid have to be considered.Rosmarinic acid (RA) is a well-known ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. In the search for enzymes involved in RA biosynthesis in the hornwort Anthoceros agrestis, the hydroxycinnamoyltransferase sequence with the highest similarity to rosmarinic acid synthase from Lamiaceae has been amplified and heterologously expressed in Escherichia coli. In parallel, the single cytochrome P450 sequence belonging to the CYP98 group in Anthoceros agrestis was isolated and expressed in Saccharomyces cerevisiae which did not result in protein formation. Codon optimization and co-expression with NADPH:cytochrome P450 reductase (CPR) from Coleus blumei resulted in the formation of active enzymes. Both, the hydroxycinnamoyltransferase and CYP98 were characterized with respect to their temperature and pH optimum as well as their substrate acceptance. The hydroxycinnamoyltransferase (AaHCT6) readily accepted p-coumaroyl- and caffeoyl-CoA with a slightly higher affinity towards p-coumaroyl-CoA. The best acceptor substrate was shikimic acid (Km 25 µM with p-coumaroyl-CoA) followed by 3-hydroxyanthranilic acid (Km 153 µM with p-coumaroyl-CoA). Another accepted substrate was 2,3-dihydroxybenzoic acid. Anthranilic acid and 4-hydroxyphenyllactic acid (as precursor for RA) were not used as substrates. p-Coumaroylesters and -amides are substrates hydroxylated by CYP98 hydroxylases. The only CYP98 sequence from Anthoceros agrestis is CYP98A147. The best substrates for the NADPH-dependent hydroxylation were p-coumaroylanthranilic and p-coumaroyl-3-hydroxyanthranilic acids while p-coumaroylshikimic and p-coumaroyl-4-hydroxyphenyllactic acids were poor substrates. The biosynthetic pathway towards rosmarinic acid thus still remains open and other enzyme classes as well as an earlier introduction of the 3-hydroxyl group to afford the caffeic acid substitution pattern must be taken into consideration.
Highlights
The model plant Anthoceros agrestis Paton (Anthocerotaceae) belonging to the hornworts is known for its accumulation of various hydroxycinnamic acid derivatives, such as rosmarinic acid (RA) andmegacerotonic acid or the alkaloid anthocerodiazonin
The conserved amino acid residues Thr and Trp located between the “arginine handle” and the DFGWG motif were reported to be involved in substrate binding and catalysis besides the catalytic His residue of the HxxxDG motif (Chiang et al 2018)
Values were calculated with the help of Michaelis–Menten (MM) and Hanes–Woolf plots (HW)
Summary
The model plant Anthoceros agrestis Paton (Anthocerotaceae) belonging to the hornworts is known for its accumulation of various hydroxycinnamic acid derivatives, such as rosmarinic acid (RA) and (hydroxy)megacerotonic acid or the alkaloid anthocerodiazonin Fig. S1) (Takeda et al 1990a; Trennheuser et al 1994) as well as numerous other hydroxycinnamic and benzoic acid derivatives (Trennheuser 1992). As such a wealth of phenolic compounds has rarely been described in mosses and liverworts, investigations into the biochemical machinery of Anthoceros and the encoding genes are interesting with regard to its evolutionary development. RA is better known as a typical compound of members of the seed plant families Lamiaceae (subfamily Nepetoideae) and Boraginaceae, its wide occurrence in other taxa of the seed plants and in ferns has been documented (Petersen et al 2009). Plectranthus scutellarioides, Solenostemon scutellarioides) (Petersen et al 1993), and verified in other plant species (Ma et al 2015)
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