Poster: Enzymology

Abs # 250: Enzymatic Formation of Pyropheophorbide: Characterization of@Pheophorbide Demethoxycarbonylase in Chlamydomonas reinhardtii

Presenter:	Furuya, Keiko , k-furuya@k3.dion.ne.jp
Authors	Furuya, Keiko  (A)   Amano, Toyoki  (A)   Shioi, Yuzo  (A)
Affiliations:	(A): Department of Biology and Geoscience, Faculty of Science, Shizuoka University

The formation of pyropheophorbide has been reported in the breakdown of chlorophylls in plants and algae. As shown in accompanying paper, we previously reported an enzyme, pheophorbidase which involves in the pyropheophorbide formation. In this study, an alternative enzyme, termed "pheophorbide demethoxycarbonylase (PDC)", was highly purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. This enzyme had produced no intermediate as in the pheophorbidase reaction, indicating that it converts pheophorbide a directly into pyropheophorbide a, probably by nucleophilic reaction. Purification of this enzyme was performed by several chromatographic steps using ion-exchange, hydrophobic, and gel filtration. PDC consisted of both senescence-induced and constitutive enzymes, but only the senescence-induced type of PDC was obtained, because quantities of constitutive PDC were too small. The molecular weight of PDC was 170,000. The K_m value against pheophorbide a was 283 mM. Methanol, which is an inhibitor of pheophorbidase, had no specific effect on PDC activity. N-ethylmaleimide inhibited the enzymatic activity to 24% of the control, suggesting a contribution of SH group. PDC activity was only detected in Chlamydomonas cells, although, no activity was found when 7 plant species lacking pheophobidase activity were tested. Substrate specificity and kinetical analyses of PDC are now underway. We also discuss that at least two alternative pathways that are catalyzed by two different enzymes, pheophorbidase and PDC, exist for the formation of pyropheophorbide a.