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

Piperonylic acid (PA) is a natural molecule bearing a methylenedioxy f unction that closely mimics the structure of trans-cinnamic acid. The CYP73A subfamily of plant P450s catalyzes trans-cinnamic acid 4-hydrox ylation, the second step of the general phenylpropanoid pathway. We sh ow that when incubated in vitro with yeast-expressed CYP73A1, PA behav es as a potent mechanism-based and quasi-irreversible inactivator of t rans-cinnamate 4-hydroxylase. Inactivation requires NADPH, is time dep endent and saturable (K-I = 17 mu M, k(inact) = 0.064 min(-1)), and re sults from the formation of a stable metabolite-P450 complex absorbing at 427 nm. The formation of this complex is reversible with substrate or other strong ligands of the enzyme. In plant microsomes PA seems t o selectively inactivate the CYP73A P450 subpopulation. It does not fo rm detectable complexes with other recombinant plant P450 enzymes. In vivo PA induces a sharp decrease in 4-coumaric acid concomitant to cin namic acid accumulation in an elicited tobacco (Nicotiana tabacum) cel l suspension. It also strongly decreases the formation of scopoletin i n tobacco leaves infected with tobacco mosaic virus.