Isolation and sequence of a cDNA encoding the Jerusalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenylpropanoid pathway.

Abstract

Cinnamate 4-hydroxylase [CA4H; trans-cinnamate,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 lignification 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 identifi'ed in this incomplete sequence, which was used as a probe for the isolation ofa 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 purified protein. The calculated pI is 9.78, in agreement with the chromatographic behavior and two-dimensional electrophoretic 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 similarity (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.

Regulation of the Cinnamate 4-Hydroxylase (CYP73A1) in Jerusalem Artichoke Tubers in Response to Wounding and Chemical Treatments

“…The second reaction of the sequence is catalyzed by a P450 called C4H. Complete cDNA sequences were recently reported for the enzymes from Jerusalem artichoke (Helianthus tuberosus; Teutsch et al, 1993), mung bean (Vigna radiata; Mizutani et al, 1993), and alfalfa (Medicago sativa; …”

“…The second reaction of the sequence is catalyzed by a P450 called C4H. Complete cDNA sequences were recently reported for the enzymes from Jerusalem artichoke (Helianthus tuberosus; Teutsch et al, 1993), mung bean (Vigna radiata; Mizutani et al, 1993), and alfalfa (Medicago sativa; posed nomenclature for P450s (Nebert et al, 1993), the H. tuberosus enzyme was termed CYP73A1. Enzymes of the general phenylpropanoid pathway are usually expressed in a coordinate manner, but the regulatory mechanisms underlying coordinated expression are unclear.…”

Subcellular Localization Studies of Three Phenylalanine Ammonia-Lyases and Cinnamate 4-Hydroxylase from <i>Scutellaria Baicalensis</i> Using GFP Fusion Proteins

“…C4H constitutes the CYP73 family of the cytochrome P450 monooxygenases that catalyze monooxygenase reactions in plants that are often involved in the biosynthesis of diverse metabolites (Chapple, 1998). C4H controls the carbon flux required for the synthesis of many phytoalexins when plants are challenged by pathogens (Teutsch et al, 1993). Therefore, PAL and C4H are important compounds involved in the regulation of biosynthesis and flux control in the S. baicalensis flavone pathway (Fig.…”

Cinnamate 4‐hydroxylase from Catharanthus roseus and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli

“…lignins, pigments and phytoalexins. The enzyme was completely purified some years ago [2], and cDNA clones were reported for a few plants (Helianthus tuberosus [3], Medicago sativa [4], Phaseolus aureus [5], and Zinnia elegans (AC U 19922). In the first part of this work we describe the molecular characterization of C4H from Madagascar periwinkle (Catharanthus roseus) and studies on the regulation in cell cultures.…”