Poster: Enzymology

Abs # 249: Enzymatic Formation of Pyropheophorbide: Purification and Cloning of Pheophorbidase from Raphanus sativus

Presenter:	Suzuki, Yasuyo , r5844001@ipc.shizuoka.ac.jp
Authors	Suzuki, Yasuyo  (A)   Amano, Toyoki  (A)   Shioi, Yuzo  (A)
Affiliations:	(A): Department of Biology and Geoscience, Faculty of Science, Shizuoka University

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 (1). In this study, one enzyme, designated gpheophorbidase (Phedase)h, was purified nearly to homogeneity from cotyledons of radish (Raphanus sativus) and cloned 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 molecular weight of both Phedases was 113,000. The K_m values against pheophorbide a for both Phedases were 14-15 mM. Both enzymes used pheophorbides a and b and bacteriopheophorbide a as the substrates, but not chlorophyll derivatives. These results indicate that the enzymes are specific for the structure of substrate, for instance, absence of Mg and phytol chain, and also a single bond at positions C-17 and C-18. The activity of both Phedases was inhibited by the reaction product, methanol but not ethanol. 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, we cloned the gene by an RT-PCR. Relationship of this enzyme and other enzymes will be discussed. 1. Suzuki, Y. and Shioi, Y. (2002) Photosyn. Res. 74: 225-233.