Cloning, sequence, expression, and characterization of protoporphyrinogen IX oxidase from chicory

Protoporphyrinogen IX oxidase (EC 1.3.3.4) is the molecular target of peroxidizing herbicides. We isolated and sequenced the cDNA for the plastidal protox from chicory (Cichorium foliosum). The gene encodes a protein of 555 amino acid residues with a calculated molecular mass of 60,244 Da. The deduced amino acid sequence exhibited high homology to the sequences from tobacco and Arabidopsis thaliana. Full-length and truncated cDNA constructs were ligated into a pQE vector, resulting in the expression of five length variants of protoporphyrinogen IX oxidase containing an N-terminal 6xHis-tag which allowed purification close to homogeneity. While the full-length recombinant enzyme was inactive, the removal of 34 or 57 amino acid residues from the N-terminus of the putative transit peptide resulted in the expression of active enzymes. The protein Variant that was reduced by 57 residues was the most active of the recombinant enzymes. The enzymatic properties of this recombinant enzyme were compared to that of the native protox, which we solubilized from chicory membranes and purified 670-fold. The recombinant protoporphyrinogen IX oxidase had a 4 times lower specific activity than the purified native enzyme. The recombinant protein was 2-10 times less sensitive to typical peroxidizing herbicides than the crude native enzyme. I-50 values of several peroxidizing compounds were determined for the native and recombinant protoporphyrinogen IX oxidase. (C) 2000 Academic Press.