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

摘要

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-132-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. nnnn--------------------------------------------------------------------------------nChanges in the color of leaves and the ripening of fruits are visible results of the breakdown of chlorophylls (Chls) due to senescence or maturation. The pathway for breakdown of Chls consists of several reaction steps (for reviews, see Hendry et al., 1987; Brown et al., 1991; Hörtensteiner, 1999; Kräutler and Matile, 1999; Matile et al., 1999; Takamiya et al., 2000), and the pathway is operationally divided into three stages. The early stage includes modification of the side chains of the tetrapyrrole macrocyclic and isocyclic rings. The middle stage involves cleavage of the macrocyclic ring by pheophorbide (Pheid) a oxygenase (PaO) and its successive modifications (Rodoni et al., 1997; Wüthrich et al., 2000; Pruinská et al., 2003). The last stage is the subsequent degradation of an open tetrapyrrole to smaller carbon- and nitrogen-containing fragments, such as organic acids, via monopyrroles (Suzuki and Shioi, 1999; Losey and Engel, 2001). nInformation concerning the enzymes involved in the early stage modification of macrocylic and isocyclic rings has gradually accumulated in recent years. The first step in the degradation of Chl a is hydrolysis of the phytyl ester linkage catalyzed by chlorophyllase (EC 3.1.1.14), which forms chlorophyllide (Chlid) a and phytol. Although the activity of chlorophyllase was revealed about 90 years ago (Willstätter and Stoll, 1913), molecular cloning of the gene was accomplished only recently (Jacob-Wilk et al., 1999; Tsuchiya et al., 1999). Based on homology searching of sequences and expression, it was determined that the coronatine-induced gene ATHCOR1 was the gene encoding chlorophyllase (Tsuchiya et al., 1999). nnA release of magnesium (Mg) from the macrocyclic ring causes the formation of Pheid a. An activity catalyzing this reaction has been reported in photosynthetic bacteria, algae, and higher plants and is considered to be due to an enzyme that has been designated Mg-dechelatase (Owens and Falkowski, 1982; Ziegler et al., 1988; Shioi et al., 1991; Vicentini et al., 1995). Previously, we demonstrated that the release of Mg2+ from Chlid a is not due to an enzyme, but to a low-molecular-mass, heat-stable substance that has been designated Mg-dechelating substance (Shioi et al., 1996a). The highly purified substance is, however, specific not only for Mg2+ but also for divalent cations. Therefore, the substance was renamed metal-chelating substance (Suzuki and Shioi, 2002). Recent studies confirmed these results, and metal-chelating substance is a possible candidate for the substance that catalyzes the Mg-dechelating reaction (Kunieda et al., 2005; Suzuki et al., 2005). nnThe final step of macrocyclic ring modification is the conversion of Pheid a to pyropheophorbide (PyroPheid) a. Two types of enzymes that catalyze alternative reactions in the formation of PyroPheid a were found (Shioi et al., 1996b; Watanabe et al., 1999; Doi et al., 2001; Suzuki et al., 2002). As shown in Figure 1, one route consists of two reactions: first, enzymatic conversion of Pheid a to a precursor of PyroPheid a, identified as C-132-carboxylPyroPheid a, and, next, spontaneous conversion of the precursor to PyroPheid a (Shioi et al., 1996b; Watanabe et al., 1999). This enzyme was designated pheophorbidase (PPD). Its activity is widely distributed but confined to some orders of higher plants, and, thus, this reaction may be specific for certain orders of plants (Suzuki et al., 2002). The other enzyme, termed Pheid demethoxycarbonylase, was partially purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii (Doi et al., 2001). This enzyme produced no intermediate, as shown in the PPD reaction, indicating that it converts Pheid a directly into PyroPheid a, probably by an acetyl transferase reaction. nnnnnnView larger version (15K):n[in this window]n[in a new window]n Figure 1. Demethoxycarbonyl reaction of Pheid a to PyroPheid a catalyzed by PPD. The reaction is composed of two steps: enzymatic conversion of Pheid a to a precursor, C-132-carboxylPyroPheid a, followed by spontaneous conversion of the precursor to PyroPheid a. The site of the enzymatic reaction is indicated by a black arrow.n n nn nPreviously, we purified PPD from Chenopodium album and the N-terminal sequence was determined (Watanabe et al., 1999); however, there is no information concerning the substrate specificity of this enzyme or the molecular structure, including gene cloning. Furthermore, little is known about the significance of this reaction step, including whether this is a true auxiliary step in vivo or whether it is a side reaction of the esterase. In this study, we purified two types of PPD from the senescent cotyledons of radish (Raphanus sativus) to homogeneity and examined their substrate specificity in a variety of Chl derivatives; we also determined N-terminal and internal amino acid sequences and the cDNA sequence. Based on the results of substrate specificity, we discuss the role of this enzyme in vivo in relation to the formation of Chl catabolites.