Substrate specificities in the chlorophyll catabolism of aquatic protists determined with <i>in vivo</i> phagocytotic assays

Chlorophylls (Chls) are ubiquitous photosynthetic pigments with inherent potential to generate cytotoxic reactive oxygen species. Therefore, all phototrophs and any phagotrophs that attempt to digest phototrophic cells have presumably developed mechanisms to mitigate this phototoxicity. In aquatic environments, the Chls produced by the dominant producers, microalgae, are catabolized into nonphototoxic pigments, cyclopheophorbide enols (CPEs), either by microalga-feeding protists or autonomously, particularly by those carrying secondary chloroplasts during the dismantling of their chloroplasts. However, the biochemistry underpinning CPE-accumulating Chl catabolism remains largely unexamined. To characterize the reactions in the transformation pathway and identify the pivotal enzyme for the formation of the seven-membered ring distinctive to CPEs, we conducted qualitative in vivo experiments using hemisynthetically prepared Chl derivatives in the cells of a euglenozoan algivorous (phycophagic) protist, Peranema trichophorum NIES-4660. We supplied polymer beads coated with Chl-b derivatives with their food cells, a unicellular red alga, Cyanidioschyzon merolae, which exclusively contains Chl-a. After administration of Chl-b or its free base with the beads, we detected a CPE derivative with a formyl group at the C7 position [cyclopheophorbide b-enol (cPPB-bE)], clearly derived from the appended derivatives, and not from the Chl-a of the alga. In contrast, cPPB-bE was not detected when zinc- and copper-metalated Chls and C132-demethoxycarbonylated Chl-b were added, although the latter resulted in the generation of its demetalated free-base form. These results indicate that (I) pheophytins are the actual substrates of the cyclization enzyme and (II) cyclization proceeds after the enzymatic dechelation of the central magnesium of natural Chls.