Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway (higher plants/plant defense/cDNA cloning/deduced amino acid sequence/molecular evolution)

Cinnamate 4-hydroxylase (CA4H; trans-cin- namate,NADPH:oxygen oxidoreductase (4-hydroxylating), EC 1.14.13.11) is a cytochrome P450 that catalyzes the first oxygenation step of the general phenylpropanoid metabolism in higher plants. The compounds formed are essential for ligni- fication and defense against predators and pathogens. We recently reported the purification of this enzyme from Mn2+- induced Jerusalem artichoke (Helianthus tuberosus L.) tuber tissues. Highly selective polyclonal antibodies raised against the purified protein were used to screen a Agtll cDNA expression library from wound-induced Jerusalem artichoke, allowing isolation of a 1130-base-pair insert. Typical P450 domains were identified in this incomplete sequence, which was used as a probe for the isolation of a 1.7-kilobase clone in a AgtlO library. A full-length open reading frame of 1515 base pairs, encoding a P450 protein of 505 residues (Mr = 57,927), was sequenced. The N terminus, essentially composed of hydrophobic residues, matches perfectly the microsequenced N terminus of the pu- rified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electropho- retic analysis of CA4H. Synthesis of the corresponding mRNA is induced in wounded plant tissues, in correlation with CA4H enzymatic activity. This P450 protein exhibits the most simi- larity (28% amino acid identity) with avocado CYP71, but also good similarity with CYP17 and CYP21, or with CYP1 and CYP2 families. According to current criteria, it qualifies as a member of a new P450 family.