DESIGN OF FLUORESCENT SUBSTRATES AND POTENT INHIBITORS OF CYP73AS, P450S THAT CATALYZE 4-HYDROXYLATION OF CINNAMIC ACID IN HIGHER-PLANTS

CYP73As are the major functional cytochromes P450 in higher plants, Se veral of them have been shown to catalyze the 4-hydroxylation of cinna mic acid, the first oxidative step in the synthesis of lignin, flavono ids, coumarins, and other phenylpropanoids. The coding sequence for CY P73A1, the enzyme from Helianthus tuberosus, has been isolated and exp ressed in yeast. Previous studies indicate that the yeast-expressed en zyme is capable of metabolizing cinnamic acid and several small, plana r molecules but with low efficiency. Using this we further examined ho w CYP73A1 could bind and metabolize a set of possible alternate substr ates. We show here that naphthalenes, quinolines, and indoles substitu ted with an aldehyde, a carboxylic, or a sulfonic acid group make good ligands and substrates for CYP73A1. The best ligands are hydroxynapht hoic acids, which show higher affinity than cinnamate, Naphthalene, 2- naphthol, and molecules with two-carbon side chains, such as natural a nd synthetic auxins, are not substrates of this enzyme. Methyl-2-napht hoate and 2-hydroxy-1-naphthoic acid are strong ligands of CYP73A1 but are not metabolized. Uncoupling and low spin conversion induced by th ese compounds suggest that their positioning in the heme pocket is ina dequate for catalysis, These compounds can act as potent inhibitors of the second step of the phenylpropanoid pathway, the first described s o far. The molecule which most closely mimics cinnamic acid, 2-naphtho ic acid, is metabolized with a catalytic turnover and efficiency simil ar to those measured with the physiological substrate, Using this comp ound we designed a fluorometric assay to measure the catalytic activit y of CYP73As, This assay was then used to monitor the CYP73As activity in microsomes from transgenic yeast and several plant species.