Piperonylic acid, a selective, mechanism-based inactivator of the trans-cinnamate 4-hydroxylase: a new tool to control the flux of metabolites in the phenylpropanoid pathway

摘要

Piperonylic acid (PA) is a natural molecule bearing a methyl-enedioxy function that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyses trans-cinnamic acid 4-hydroxylation, the second step of the general phenylpropanoid pathway. When incubated in vitro with yeast-expressed CYP73A1, PA behaved as a potent mechanism-based and quasi-irreversible inactivator of trans-cinnamate 4-hydroxylase. Inactivation required NADPH, was time dependent and saturable, and resulted from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex was reversible with substrate or other strong ligands of the enzyme. In Jerusalem artichoke (Helianthus tuberosus) tuber microsomes PA seemed to selectively inactivate the CYP73A P450 subpopulation. It did not form detectable complexes with other recombinant plant P450 enzymes. In vivo PA induced a sharp decrease in 4-coumaric acid concomitant to cinnamic acid accumulation in an elicitedtobacco (Nicotiana tabacum) cell suspension. It also strongly decreased the formation of scopoletin in 3-week-old tobacco leaves infected with tobacco mosaic virus [tobacco mosaic tobamovirus].