Abstract
Black pepper (Piper nigrum) is among the world’s most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.
Highlights
Plants are known for their remarkable structural diversity of specialized metabolites [1]
The resulting plethora of compounds is partly based on the evolution of cytochrome P450 enzymes (CYPs), heme-dependent monooxygenases that catalyze a wide variety of hydroxylation and monooxygenation reactions [2,3]
The characteristic methylenedioxy bridge of the phenylpropanoid part of the molecule is one of the unsolved biosynthetic hallmarks in piperine formation. It can be introduced at the level of ferulic or feruperic acid, and late as a final modification after amide bond formation at the level of feruperine Based on a transcriptional approach, using RNA sequencing data from different black pepper organs, combined with functional expression in yeast, we describe the identification and partial characterization of a fruit specific cytochrome P450 enzyme that catalyzes the formation of the 3,4-methylenedioxy group in the aromatic part of the piperine molecule
Summary
Plants are known for their remarkable structural diversity of specialized metabolites [1]. Eukaryotic CYPs show an N-terminal membrane anchor and are known as ER-associated proteins of many biochemical pathways. They obtain electrons from NADPH-cytochrome P450 reductases (CPRs) usually required for catalytic activity. Besides common reactions like oxidation and hydroxylation of a carbon skeleton, CYPs are known for unusual reactions including oxidative C-C bond cleavage, methylenedioxy bridge formation, and phenol coupling [5]. Methylenedioxy bridges and phenolcouplings can be performed by the CYP719 clade within the heterogeneous CYP71 clan, classified as CYP719A [6,7], whereas phenol-coupling is performed by the CYP719B subclade, respectively [8].
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