The role of hydroxycinnamoyltransferases and CYP98s in shaping the phenolic profile of Sarcandra glabra

In the development of angiosperms, the Chloranthaceae are placed at a branching point, since the family already belongs to the angiosperms but is still situated together with the Magnoliids before the switch that later separates monocotyledons from dicotyledons. Sarcandra glabra, which is native to South Asia, is one of a few members of the Chloranthaceae, and used in Traditional Chinese Medicine. The anti-inflammatory and antioxidant effects of the herbal drug have been studied in animal models and have been linked to the presence of various natural products. Among the at least twenty known phenolic substances are rosmarinic acid (RA), chlorogenic acid (CA) and caffeoyl-5-O-shikimic acid as well as their derivatives. CA, caffeoyl-5-O-shikimic acid and RA are produced in plants by the esterification of 4-coumaric acid with quinic acid, shikimic acid or 4-hydroxyphenyllactic acid, followed by one or two hydroxylations in meta-position, respectively. The former reaction is catalyzed by a hydroxycinnamoyltransferase (HCT) from the BAHD superfamily, whereas the hydroxyl moieties are introduced by CYP98. In the present study four HCTs and one CYP98 from Sarcandra glabra were characterized in detail with regard to their catalytic properties in vitro. SgHST showed the highest catalytic efficiency among the HCTs with p-coumaroyl-CoA and a constant concentration of shikimic acid. It is therefore assumed that SgHST is mainly responsible for the formation of p-coumaroyl-5-O-shikimic acid despite its low expression. Furthermore, high affinity to 3-hydroxyanthranilic acid was found, of which the in vivo significance is unclear. SgHQT1 was able to catalyze the acylation of quinic acid with high catalytic efficiency, leading to direct formation of CA or the precursor p-coumaroyl-5-O-quinic acid. Interestingly, the acylation of all three hydroxyl moieties of shikimic acid was observed. Additionally, SgHQT1 was able to transfer a p-coumaroyl moiety to a wide range of (di)hydroxybenzoic acid derivatives, indicating a relaxed selectivity. In contrast, SgHQT2 catalyzed the formation of cryptochlorogenic acid (cCA) and its precursor regiospecifically. The low catalytic efficiency and low transcript levels in the plant parts of Sarcandra glabra justify the low abundance of cCA. Furthermore, tests with SgHQT2 demonstrated the synthesis of caffeoyl-4-O-shikimic acid by acylation of shikimic acid at C4-OH. The formation of the RA precursor p-coumaroyl-4’-hydroxyphenyllactic acid was observed in enzyme tests with SgRAS. The preferential conversion of p-coumaroyl-CoA over caffeoyl-CoA and 4-hydroxyphenyllactic acid over 3,4-dihydroxyphenyllactic acid was derived from the catalytic efficiencies of the respective combinations. Investigations of SgCYP98A235 showed the enzymatic hydroxylation in meta-position of precursors of RA, CA, cCA and caffeoyl-5-O-shikimic acid under consumption of NADPH and O2. Both, caffeoyl-4’-hydroxyphenyllactic acid and p-coumaroyl-3’,4’-dihydroxyphenyllactic acid were converted to RA by hydroxylation of the respective moiety. Biochemical characterization revealed a high affinity for p-coumaroyl-4’-hydroxyphenyllactic acid and thus a preferential C3-hydroxylation. High transcript levels of SgRAS and SgCYP98A235 were made accountable for RA concentrations of up to 3.6 % of the plant dry weight. Further isoforms of HCTs and CYP98s were investigated, whereby in tests with SgHCT-F an involvement in the biosynthesis of benzyl 2-β-glucopyranosyloxybenzoate was expected, as well as the hydroxylation of 4-coumaric acid esters by SgCYP98A234, neither of which could be confirmed. Independent studies have suggested the involvement of [RAS] Cluster in the formation of RA, but in addition to the lack of typical conserved amino acid motifs of HCTs, no catalytic activity was observed in enzyme assays. Overall, this work demonstrates the involvement of SgHCTs and SgCYP98s in phenolic metabolism in Sarcandra glabra by identification and isolation of the coding sequences, heterologous synthesis of the proteins and biochemical characterization of the respective enzymes. In particular, SgHQT2 was shown to be a key enzyme in the selective formation of cCA. In addition, different relative transcription rates of the enzymes in eight plant parts showed a correlation with the content of phenolic compounds. The biosynthesis of RA, CA, and caffeoyl-5-O-shikimic acid in Sarcandra glabra was proven to be analogous to already established theories in the most important points, despite the evolutionary distance of the Chloranthaceae to other angiosperms. However, phylogenetic studies of SgRAS suggest a common evolutionary ancestor of the enzyme with hydroxycinnamoyl-CoA:spermine/spermidine hydroxycinnamoyltransferases. The findings obtained here represent a piece of the puzzle that further completes the big picture of specialized plant metabolism.