Piper nigrum CYP719A37 Catalyzes the Decisive Methylenedioxy Bridge Formation in Piperine Biosynthesis

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.

Exploring the Chemical Space of Kawakawa Leaf (Piper excelsum)

“…Interestingly these two compounds both feature triple modifications to the aromatic ring: three methoxy groups in the case of elemicin; a methylenedioxy bridge and a methoxy group for myristicin. Piperic acid is synthesised in P. nigrum by the enzyme CYP719A37, which forms the methylenedioxy bridge [ 27 ]. This enzyme has considerable selectivity for vanilloid precursors with an aliphatic chain length of five carbons or more.…”

Cytochrome P450 Enzymes as Key Drivers of Alkaloid Chemical Diversification in Plants

“…( S )-Scoulerine is also subject to another methoxyphenol cyclization catalyzed by CYP719A5 in California poppy ( Ikezawa et al, 2009 ), CYP719A14 in Mexican prickly poppy ( Díaz Chávez et al, 2011 ), or CYP719A25 in opium poppy ( Desgagné-Penix et al, 2010 ; Dang and Facchini, 2014b ) to form ( S )-cheinlanthifoline. Recently, CYP719A37 in black pepper ( Piper nigrum ) has been found to be responsible for the presence of the methylenedioxy bridge in piperic acid, a precursor of the pungent alkaloid piperine ( Schnabel et al, 2021 ).…”

The terminal enzymatic step in piperine biosynthesis is co‐localized with the product piperine in specialized cells of black pepper (Piper nigrum L.)

“…Subsequently, they described a piperine synthase (PS) activity from shoots of black pepper, rather than from fruits (Geisler & Gross, 1990). By a differential RNA sequencing (RNA‐Seq) approach comparing various black pepper organs, we recently identified the transcripts encoding enzymes for three sequential piperine biosynthetic steps in black pepper fruits (Schnabel et al., 2020; Schnabel, Athmer, et al., 2021; Schnabel, Cotinguiba, et al., 2021). CYP719A37, a cytochrome P450 monooxygenase, catalyzes the formation of the 3,4‐methylenedioxy group from feruperic acid to piperic acid, followed by an activating piperic acid CoA ligase and a terminal BAHD‐type acyltransferase, termed PS, that catalyzes the amide bond formation between the piperoyl CoA‐ester and the piperidine heterocycle towards piperine (Figure 1).…”