Identification and characterization of piperine synthase from black pepper, Piper nigrum L.

Abstract

Black pepper (Piper nigrum L.) is the world’s most popular spice and is also used as an ingredient in traditional medicine. Its pungent perception is due to the interaction of its major compound, piperine (1-piperoyl-piperidine) with the human TRPV-1 or vanilloid receptor. We now identify the hitherto concealed enzymatic formation of piperine from piperoyl coenzyme A and piperidine based on a differential RNA-Seq approach from developing black pepper fruits. This enzyme is described as piperine synthase (piperoyl-CoA:piperidine piperoyl transferase) and is a member of the BAHD-type of acyltransferases encoded by a gene that is preferentially expressed in immature fruits. A second BAHD-type enzyme, also highly expressed in immature black pepper fruits, has a rather promiscuous substrate specificity, combining diverse CoA-esters with aliphatic and aromatic amines with similar efficiencies, and was termed piperamide synthase. Recombinant piperine and piperamide synthases are members of a small gene family in black pepper. They can be used to facilitate the microbial production of a broad range of medicinally relevant aliphatic and aromatic piperamides based on a wide array of CoA-donors and amine-derived acceptors, offering widespread applications.

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

“…Even then, no signal was ever observed in leaves or flowers. In summary, the Western blot results are consistent with rising piperine levels and the transcriptome data that suggest highest transcript abundance in fruits around 60 dpa (Schnabel, Athmer, et al., 2021). It is important to note that neither unspecific signals indicating no protein degradation in the optimized extraction protocol nor any cross‐reactivity show up on any of the Western blots, consistent with an apparent high specificity of the purified polyclonal PAS‐AB1 antibody against PS/PAS.…”

“…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).…”

“…The PS acyltransferase in P. nigrum is part of a small gene family of similar BAHD‐type acyltransferases that were identified in the black pepper genome (Hu et al., 2019). A second acyltransferase, termed piperamide synthase (PAS), with 63% identity at the amino acid level to PS, was also identified to be co‐expressed in fruits with equally high transcript levels (Schnabel, Athmer, et al., 2021). In contrast to PS, PAS, at least in vitro , preferentially produces piperine isomers that are not detected in extracts of freshly harvested fruits, and therefore the role of PAS in the fruits remains obscure.…”

“…In contrast to PS, PAS, at least in vitro , preferentially produces piperine isomers that are not detected in extracts of freshly harvested fruits, and therefore the role of PAS in the fruits remains obscure. Compared to PS, the enzyme displays a much higher substrate promiscuity in vitro (Schnabel, Athmer, et al., 2021).…”

Metabolomics Insights into Chemical Convergence in Xanthomonas perforans and Metabolic Changes Following Treatment with the Small Molecule Carvacrol

“…The other plant metabolites identified in X. perforans include piperidine, the parent backbone for piperine in Piper nigrum, and enterodiol sulfate, an alkaloid found in flax seeds [64,65,67]. A Xanthomonas species, X. campestris pv.…”