Piperamide biosynthesis in black pepper (Piper nigrum)
Final Report Abstract
Supported by a differential RNA-Seq approach of different black pepper (Piper nigrum) organs and the synthesis of potential precursors and substrates, three decisive steps in piperine biosynthesis and the reaction sequence of these reactions were established. Starting with feruperic acid, a C2-extended ferulic acid molecule, a characteristic methylenedioxy bridge is introduced into the aromatic part of piperine by a cytochrome P450-dependent monooxygenase, piperic acid synthase (CYP719A37) that is specific for feruperic acid. The enzyme does not accept ferulic acid or any other natural compound that contains the essential aromatic vanilloid-structure. A corresponding reductase from P. nigrum was also cloned and functionally expressed in parallel. The enzyme is not expressed specifically in the fruits. The resulting piperic acid is activated by a specific piperoyl CoA-ligase. Piperine synthase, a BAHD-like acyltransferase subsequently catalyzes the final amide formation with piperidine as the amine donor resulting in piperine. Besides piperine synthase, a second enzyme, termed piperamide synthase was also cloned from ripening fruits and functionally expressed. The recombinant enzyme has a promiscuous substrate specificity and catalyzes the efficient amide bond formation from a wide variety of aliphatic and aromatic CoA-esters with different amines. Using piperoyl CoA and piperidine as substrates, this enzyme preferentially produces different piperine isomers, compounds that are not present in freshly extracted black pepper fruits. This unusual properties might be due to differences of in vivo conditions, as compared to in vitro enzyme assays. Crystallization trials have been initiated to identify the amino acids that are responsible for the observed differences in substrate specificity. Determination of the protein structure of piperine synthase will be essential to optimize both synthases for production of relevant amides in biotechnology and pharmacy. Immunodetection of piperine and piperamide synthase by polyclonal antibodies, western blots and immunolocalization by fluorescent secondary antibodies are consistent with imaging of piperine by fluorescence microscopy and MS-imaging data. These data suggest that piperine synthase and presumably all reactions of piperine biosynthesis are localized in specialized cells of the fruit perisperm of ripening black pepper fruits.
Publications
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(2020) A piperic acid CoA ligase produces a putative precursor of piperine, the pungent principle from black pepper fruits. The Plant Journal 102, 569-581
Schnabel A, Cotinguiba F, Athmer B, Yang C, Westermann B, Schaks A, Porzel A, Brandt W, Schumacher F and Vogt T
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(2021) Identification and characterization of piperine synthase from black pepper, Piper nigrum L. Commun. Biol 4, 445
Schnabel, A., Athmer, B., Manke, K., Schumacher, F., Cotinguiba, F., Vogt T
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(2021) Piper nigrum CYP719A37 catalyzes the decisive methylenedioxy bridge formation in piperine biosynthesis. Plants 10, 128
Schnabel A, Cotinguiba F, Athmer B and Vogt T
