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

Abstract

Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Piper nigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with a blend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzes the reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co-localization of enzymes and products, but also potential transport of piperamides to the sink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), provides experimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit perisperm. PS accumulates during early stages of fruit development and its level declines before the fruits are fully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by its strong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasing numbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells when monitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperine and additional piperamides were also detected in cells distributed in the cortex of black pepper roots. In summary, the data provide comprehensive experimental evidence of and insights into cell-specific biosynthesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination of fluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cells of the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthases shows that enzymes are co-localized with high concentrations of products in these idioblasts.

Comparative metabolite analysis of Piper sarmentosum organs approached by LC–MS-based metabolic profiling

“…The accumulation of these compounds was found in the roots where they may serve as defense against feeding and pathogens. Significant levels of similar piperamides were recently also detected in cells distributed in the cortex of black pepper roots [ 8 ]. Several plants, including Piper species such as P. puberulum and P. flaviflorum , have been reported to contain lignanamides [ 40 , 41 ].…”

“…Also, it remains yet unresolved to what extent they are produced independently in the two organs or transported. However, if in analogy to P. nigrum where special cells perform the piperamide synthesis, they likely are produced independently in both organs [ 8 ]. In any case, our finding shows, that separate harvesting and utilization of P. sarmentosum organs is relevant to the composition of any concoction thereof, be it for food or medicinal purposes.…”

“…Some of such applications were pharmacologically proven [ 6 ]. The most famous representative of this genus is Piper nigrum L., the well-known black pepper (The "pepper") used as spice and seasoning [ 7 , 8 ].…”

Chemical Synthesis of Trans 8-Methyl-6-Nonenoyl-CoA and Functional Expression Unravel Capsaicin Synthase Activity Encoded by the Pun1 Locus

“…Apparent K m values for the substrates of capsaicin synthase are in the high three-digit micromolar range, in the same order of magnitude as the piperine synthase that produces a similar blend of amides [ 16 ] but more than 10-fold higher than the data reported for other recombinant BAHDs [ 14 , 15 ]. These surprisingly low affinities compared to other BAHD-type acyltransferases, specifically in the case of vanilloylamine, suggests that either high substrate concentrations are present in the cells where capsaicin biosynthesis takes place, as reported for piperine synthase [ 20 ], or that in vitro assay conditions are less optimal compared to the metabolic channeling proposed for specialized cells in vivo [ 21 , 22 , 23 ]. A rather high in vivo efficiency is consistent with the reported cell-specific accumulation of capsaicin during the rather short time frame of fruit development, resulting in concentrations of up to 10 g/kg of the dry weight of these alkaloids in the fruits of chili pepper [ 24 ].…”

Molecular Aspects of Piperine in Signaling Pathways Associated with Inflammation in Head and Neck Cancer

“…This compound is biosynthesized from L-lysine and the precursor cinnamoyl-CoA as a secondary metabolite. After a series of reactions, the bioactive compound piperine is obtained from piperonyl-CoA (Figure 1) [11,12]. The attention given to the study of this molecule is mainly due to its biological properties, such as anti-inflammatory, antioxidant, immunomodulatory and anticancer properties, which allow this compound to chemically interact with various molecular targets [7].…”