Characterization and Cloning of the Chlorophyll-Degrading Enzyme Pheophorbidase from Cotyledons of Radish

Abstract

Enzymatic removal of the methoxycarbonyl group of pheophorbide (Pheid) a in chlorophyll degradation was investigated in cotyledons of radish (Raphanus sativus). The enzyme pheophorbidase (PPD) catalyzes the conversion of Pheid a to a precursor of pyropheophorbide (PyroPheid), C-13 2 -carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated nonenzymatically to yield PyroPheid a. PPD activity sharply increased with the progression of senescence in radish, suggesting de novo synthesis of PPD. The enzyme activity was separated into two peaks in anion-exchange and hydrophobic chromatography; the terms type 1 and type 2 were applied according to the order of elution of these enzymes in anion-exchange chromatography. PPD types 1 and 2 were purified 9,999-and 6,476-fold, with a yield of 0.703% and 2.73%, respectively. Among 12 substrates tested, both enzymes were extremely specific for Pheids of the dihydroporphyrin and tetrahydroporphyrin types, indicating that they are responsible for the formation of these PyroPheids. Both PPDs had molecular masses of 113,000 kD on gel filtration and showed three bands of 16.8, 15.9, and 11.8 kD by SDS-PAGE. The partial N-terminal amino acid sequences for these bands of PPD (type 2) were determined. Based on their N-terminal amino acid sequences, a full-length cDNA of PPD was cloned. The molecular structure of PPD, particularly the molecular mass and subunit structure, is discussed in relation to the results of SDS-PAGE.

The ABC Transporter PXA1 and Peroxisomal β-Oxidation Are Vital for Metabolism in Mature Leaves ofArabidopsisduring Extended Darkness

“…Results obtained with plants that express a PAO antisense construct and a pao T-DNA insertion mutant indicate strong phototoxic properties of PhA, since plants with a decreased level of PAO expression accumulate high levels of PhA in dark-treated leaves and show lethal bleaching after transfer into the light (Tanaka et al, 2003;Pruzinská et al, 2005). In white goosefoot (Chenopodium album) and radish (Raphanus sativus), there is evidence that PhA degradation also occurs via pheophorbidase activity, an enzyme methylating PhA (Shioi et al, 1996;Suzuki et al, 2006). However, in Arabidopsis, this enzyme has also been linked to methylation of primary fluorescent chlorophyll catabolite, a product downstream of PAO activity in chloroplasts (Pruzinská et al, 2007).…”

Update on the biochemistry of chlorophyll breakdown

“…However, to date only the enzyme hydrolyzing the C13 2 -methylester has been identified at the molecular level. C13 2 -demethylated catabolites have so far only been identified in the Brassicaceae, and in the past an enzyme termed pheophorbidase has been described, which is capable of hydrolyzing the C13 2 -methylester of Pheide (Suzuki et al 2006). The product of the reaction, C13 2 -carboxyl pyropheophorbide, was shown to spontaneously decarboxylate to pyropheophorbide (Shioi et al 1996b), a proposed product of Chl breakdown mainly found in algae and during post harvest senescence (Ziegler et al 1988;Aiamla-or et al 2010).…”

“…The product of the reaction, C13 2 -carboxyl pyropheophorbide, was shown to spontaneously decarboxylate to pyropheophorbide (Shioi et al 1996b), a proposed product of Chl breakdown mainly found in algae and during post harvest senescence (Ziegler et al 1988;Aiamla-or et al 2010). Pheophorbidase was cloned from Raphanus sativus (Suzuki et al 2006); it is a serine-type esterase and is highly similar to the members of the Arabidopsis methyl esterase (MES) protein family. In a recent study , recombinant MES16 was shown to hydrolyze the C13 2 methylester of both Pheide a and pFCC (or epi-pFCC), but not of a NCC.…”

Tetrapyrrole Metabolism inArabidopsis thaliana

“…At the present time, it is not clear how pyropheophorbide is further metabolized. The pheophorbidase enzyme is identified with radish (Raphanus sativus) (Suzuki et al 2006) and homologues are found in the Arabidopsis genome (At4g16690) and other plant species including Capsella rubella and two Brassica species. The distribution of homologues in a limited number of species is consistent with the findings that pheophorbidase activity is detected with the Brassicaceae and a few other species (Suzuki et al 2002b).…”