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

Protoporphyrinogen IX oxidase (EC 1.3.3.4) is the molecular target of perox idizing herbicides. We isolated and sequenced the cDNA for the plastidal pr otox from chicory (Cichorium foliosum). The gene encodes a protein of 555 a mino acid residues with a calculated molecular mass of 60,244 Da. The deduc ed amino acid sequence exhibited high homology to the sequences from tobacc o and Arabidopsis thaliana. Full-length and truncated cDNA constructs were ligated into a pQE vector, resulting in the expression of five length varia nts of protoporphyrinogen IX oxidase containing an N-terminal 6xHis-tag whi ch allowed purification close to homogeneity. While the full-length recombi nant 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 o f active enzymes. The protein Variant that was reduced by 57 residues was t he most active of the recombinant enzymes. The enzymatic properties of this recombinant enzyme were compared to that of the native protox, which we so lubilized from chicory membranes and purified 670-fold. The recombinant pro toporphyrinogen IX oxidase had a 4 times lower specific activity than the p urified native enzyme. The recombinant protein was 2-10 times less sensitiv e to typical peroxidizing herbicides than the crude native enzyme. I-50 val ues of several peroxidizing compounds were determined for the native and re combinant protoporphyrinogen IX oxidase. (C) 2000 Academic Press.