Abstract
The demethoxycarbonyl reaction of pheophorbide a in plants was investigated. Two types of enzyme that catalyze alternative reactions in the formation of pyropheophorbide a were found. In this study, one enzyme, designated pheophorbidase (Phedase), was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus) and cloning the gene. This enzyme catalyzes the conversion of pheophorbide a to a precursor of pyropheophorbide a, C-132-carboxylpyropheophorbide a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield pyropheophorbide a. Phedase consisted of both senescence-induced and constitutive enzymes and were separated by DEAE-Toyopearl. The purified enzyme showed three bands of 16.8, 15.9 and 11.8 kDa on SDS-PAGE. The partial N-terminal amino acid sequences for three bands of purified constitutive Phedase could be determined. Based on their N-terminal amino acid sequences, cDNA of Phedase was cloned by a combination of RT-PCR and RACE.
