Molecular and biochemical investigations of genes and enzymes involved in the phenolic metabolism of the hornwort Anthoceros agrestis
An important group of plant compounds are phenolics, leading to many different secondary metabolites, e.g. flavonoids, lignans and lignin, cutin and suberin as well as hydroxycinnamic acid esters and amides (e.g. chlorogenic acid and rosmarinic acid). They can act for example as natural sun screens...
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|An important group of plant compounds are phenolics, leading to many different secondary metabolites, e.g. flavonoids, lignans and lignin, cutin and suberin as well as hydroxycinnamic acid esters and amides (e.g. chlorogenic acid and rosmarinic acid). They can act for example as natural sun screens or cell wall reinforcements, essential for the conquering of dry habitats . Until now, hornworts were the only gap amongst the bryophytes in understanding the phenylpropanoid metabolism on a genomic level [2, 3]. Cinnamic acid 4-hydroxylase (C4H) from Anthoceros agrestis was heterologously expressed in Escherichia coli and Physcomitrella patens. For expression in E. coli AaC4H was fused to AaCPR, a NADPH:cytochrome P450 reductase. The resulting fusion protein produced only a small amount of 4-coumaric acid. Additionally, AaCPR was present as a soluble protein and was therefore characterized after purification by metal chelate chromatography. P. patens was introduced as a suitable expression host for plant P450s. In real time PCR experiments it was shown, that expression of AaC4H was between 270 to 3700-fold compared to two potential C4Hs from P. patens. Protein extracts from transformed cultures revealed the formation of double to triple the amount of 4-coumaric acid and increased the affinity for cinnamic acid compared to the wild-type control. Two CoA-ligases, activating different (hydroxy)cinnamic and (hydroxy)benzoic acid derivatives, were found with 4-coumarate CoA-ligase (Aa4CL) and 4-hydroxybenzoate CoA-ligase (Aa4HBCL). Both differed substantially in their substrate preference. Aa4CL accepted 4-coumaric, caffeic, cinnamic, ferulic, isoferulic, 2-coumaric and 3-coumaric acid, but lacked affinity for sinapic acid and benzoic acid derivatives. The other CoA-ligase preferably activated benzoic acid or monohydroxylated benzoic acids and several other benzoic acid derivatives (except for salicylic acid, 3-aminosalicylic acid and vanillic acid) but also demonstrated activity towards cinnamic, 2-coumaric and 3-coumaric, 4-coumaric, caffeic and isoferulic acid.
Besides Aa4CL and Aa4HBCL a third potential CoA-ligase was expressed in E. coli. The amino acid sequence of Aa20832 revealed a peroxisomal signal sequence (PTS1) and two potential transmembrane helices were identified by secondary structure prediction.The protein did not activate any tested (hydroxy)cinnamic or (hydroxy)benzoic acids but demonstrated a high ATPase activity. Aa20832 might have an activity towards fatty acids, but this was not clearly proven. Another cytochrome P450 characterized in this work was AaCYP98, a hydroxycinnamoyl ester/amide 3-hydroxylase. The native AaCYP98 was expressed in P. patens and a codon optimized sequence was expressed in S. cerevisiae. AaCYP98 was able to hydroxylate 4-coumaroyl-3'-hydroxyanthranilic acid, 4-coumaroylanthranilic acid, 4-coumaroyltyramine, 4-coumaroylshikimic acid and 4-coumaroyl-4'-hydroxyphenyllactic acid but did not accept 4-coumaroylquinic acid, 4-coumaroyl-2'-threonic acid and caffeoyl-4'-hydroxyphenyllactic acid. Since these substrates were not commercially available most of them were either isolated from plant cell cultures or synthesized enzymatically or chemically. Activity of AaCYP98 was increased 13-fold after coexpression with a CPR from Coleus blumei. At last, the cytochrome P450 AaAp626 was expressed in P. patens. In a BLASTp search, the sequence demonstrated the highest identities towards putative flavonoid 3'-hydroxylases and CYP71A1. No activity was observed in enzyme assays with 4-coumaroyl-4'-hydroxyphenyllactic acid and the three flavonoids galangin, kaempferol and naringenin. Thus, the function of this protein remains unclear. In summary, based on this work the last two enzymes of the core phenylpropanoid pathway (AaC4H and Aa4CL) and enzymes of subsequent biosynthetic pathways of the hornwort Anthoceros agrestis (AaCYP98 and Aa4HBCL), as well as a NADPH-dependent CPR (AaCPR) were identified and characterized. Moreover, two proteins with yet unknown function, a P450 and another CoA-ligase, were heterologously expressed in P. patens and E. coli. These results provide a good foundation to gain more insight into the function and evolution of the plant phenylpropanoid biosynthetic pathway.
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