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First published online December 29, 2005; 10.1104/pp.105.071290 Plant Physiology 140:716-725 (2006) © 2006 American Society of Plant Biologists Characterization and Cloning of the Chlorophyll-Degrading Enzyme Pheophorbidase from Cotyledons of Radish1Department of Biological Science, Faculty of Science, Shizuoka University, Shizuoka 422–8529, Japan
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.
Changes 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; Pru inská 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).
Information 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
A 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
The 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
Previously, 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.
Enzyme Activities and Chl Breakdown during Senescence
The concentrations of Chl in the senescent cotyledons of radish were followed spectrophotometrically after extraction (Fig. 2). Chl concentrations gradually decreased as the duration of senescence increased and reached 44% after 3 d. In contrast, PPD activity sharply increased at 1 d, was maintained at a stationary level up to 3 d, and then decreased. The activity in senescent leaves at the first day increased about 7 times compared to that of nonsenescent leaves. This was not due to an increase in the activity itself, but to de novo synthesis of the enzyme in accordance with the results of inhibitor studies using cyclohexamide. This finding is also confirmed by immunoblot analysis using a polyclonal antibody raised against a purified recombinant PPD, although several minor faint bands appeared (data not shown). Pigment analysis using HPLC showed that the substrate and product of this enzyme, Pheid and PyroPheid species, were not detected in the senescent cotyledons of radish (Suzuki and Shioi, 2004
Purification of Type 1 and Type 2 PPDs
The crude enzyme, extracted from about 5 kg (fresh weight) of 1-d-senescent cotyledons of radish, was purified by four steps of successive chromatography (Table I). The enzyme activity was separated into two peaks by chromatography on DEAE-Toyopearl and butyl-Toyopearl. These peaks are not artifacts during purification, but probably isomers, because they were reproducibly observed in one-step purification with a simple extraction followed by chromatography. The PPDs were operationally termed type 1 and type 2 according to their order of elution in DEAE-Toyopearl chromatography (Fig. 3). These enzymes increased to high levels during senescence, as seen from the results of the time-dependent study (Fig. 2), indicating that these PPDs are induced in parallel by senescence. In comparison with the activity at 1-d senescence (Fig. 3), the total activity of type 1 enzyme was about 5 times higher than that of nonsenescence, although type 2 stayed about 3 times higher. These enzymes were purified separately using chromatography. PPD types 1 and 2 were purified 9,999- and 6,476-fold with a yield of 0.703% and 2.73%, respectively. A summary of the purification of the two PPDs is presented in Table I. Some enzymatic properties (e.g. optimal pH and the effect of reaction products) were reported previously (Suzuki et al., 2002
SDS-PAGE was performed with purified PPD type 2 (Fig. 4). In non-heat-treated samples, one major band whose size was about 77 kD appeared in 15% polyacrylamide gel (Fig. 4, lane 1), while on 12% gel a single band emerged at the size of 61.3 kD. Even after 1 min of heat treatment of the enzyme in the sample buffer containing SDS and 2-mercaptoethanol, PPD was separated into three bands with molecular masses of 16.8, 15.9, and 11.8 kD (Fig. 4, lane 2). The pattern of SDS-PAGE was not changed after heating for 1 to 20 min. Similar results were also obtained for PPD type 1.
Substrate Specificity of PPD The Km for Pheid a of the two enzymes was determined in the concentration range from 1 to 40 µM. The values obtained were 14.1 and 15.1 µM for types 1 and 2, respectively (see Table II).
To determine the substrate specificity of the enzyme, several Chl derivatives were examined under standard assay conditions. As shown in Table II, both enzymes used Pheid a/b and bacterio-Pheid a as substrates, but not other Chl derivatives, such as protoChlid a, pheophytin c, Chl a/b, Chlid a, pheophytin a, and bacterio-Chl a. These results indicate that the enzymes are specific for the structure of the substrate, (e.g. the absence of Mg and a phytol chain) and also the existence of a single bond at the position of C17-C18. The Km values of the two enzymes as active substrates were calculated to be about 240 and 40 µM for Pheid b and bacterio-Pheid a, respectively. The Km value for Pheid b was the highest among the three Pheid species, suggesting that a methyl group at C7 confers a greater affinity than a formyl group. Furthermore, the Chl catabolite, nonfluorescent Chl catabolite (NCC), was examined to determine whether PPD uses it as a substrate or not. Cj-NCC, prepared from Cercidiphyllum japonicum, was kindly provided by Professor Bernhard Kräutler. No change in the HPLC peak of Cj-NCC at a retention time of 70.2 min was observed, indicating that PPD did not recognize Cj-NCC as a substrate. To test the esterase activity of this enzyme, several nitrophenyl compounds, such as nitrophenyl acetate and nitrophenyl butylate, which are used for general esterase assays, were used as the substrates. However, little or no activity was observed for these substrates, indicating that the PPD activity is not a side reaction of an esterase activity as far as the substrates tested.
Analyses of the N-terminal sequences were performed with the three peptides separated by SDS-PAGE after heat treatment of the purified PPD type 2 (Fig. 4). The amino acid sequences of three peptides were determined: 16.8 kD, EEDIWEYIYGEGADKPPTGVLMKEEFFRRY; 15.9 kD, EDIWEYIYGEGADKPPTGVLMKEEFFRHYY; and 11.8 kD, DDSVVHFVFFHGASHGAAWYYKPTTTLV (Fig. 5A). In the 15.9-kD peptide, only one amino acid of the N-terminal sequence was missing as compared to that of the 16.8 kD, although the C terminus is unknown. Two different partial amino acid sequences were thus revealed from these analyses.
A BLAST search (BLASTp; Altschul et al., 1997 ) was performed against the National Center for Biotechnology Information (NCBI) databases with the two amino acid sequences. The results showed that the protein NP_193402 of Arabidopsis (Arabidopsis thaliana) had homologies of 77% and 64% to PPD peptides, 16.8 and 11.8 kD, respectively. From the comparison with NP_193402, the amino acid sequence of 11.8 kD was located upstream from those of 16.8 and 15.9 kD. Sense and antisense degenerate primers for reverse transcription (RT)-PCR were therefore designed from the amino acid sequence of PPD, which had been constructed based on the sequences of the 11.8- and 16.8-kD peptides. From the results of RT-PCR and 3'-RACE, the partial cDNA of PPD that corresponds to the 862 bp containing the untranslated region was sequenced. The translated region of PPD had 84% identity to At4g16690, which is the cDNA of NP_193402. We assumed that the 5' ends of these mRNAs are conserved between PPD and At4g16690. To obtain the full sequence-coding region of PPD, an At4g16690-specific sense primer and a PPD-specific antisense primer containing the initiation and termination codons, respectively, were then designed. Finally, the 792-bp cDNA fragment from the initiation codon, ATG, to the termination codon, TGA, was cloned by a combination of PCRs with the template synthesized from radish RNA (Fig. 5B). We named this gene RsPPD. The nucleotide sequence reported in this article has been submitted to the DNA Data Bank of Japan (DDBJ; accession no. AB218276). Genomic Southern analysis for RsPPD showed a major band with one or two minor bands, depending on the restriction enzymes used, suggesting that RsPPD exists as multiple copies on the Raphanus genome.
The amino acid sequence deduced from the nucleic acid sequence was 263 amino acid residues corresponding to a Mr of 28,974. The Mr and subunit structure are discussed below. The deduced polypeptide has a lipase domain (Fig. 5B, underline) that was presumed to be an active site of esterases, as in the case of chlorophyllase (Tsuchiya et al., 2003
Homology searching against the deduced amino acid sequence of PPD was performed with BLAST programs using the NCBI databases GenomeNet BLAST2 and The Arabidopsis Information Resource (TAIR) BLAST 2.0. We operationally named the homologs of PPD using the prefix of their species name to distinguish the sources (i.e. RsPPD [R. sativus PPD]). From the results of the homology search, two homologs, NP_193402 from Arabidopsis (GI: 15235844, AtPPD) and CAC82615 from Capsella rubella (GI: 15866583, CrPPD), were found by BLASTp, and four homologs, CD819739 and CD818940 from Brassica napus (GI: 32501679 and GI: 32500880, BnPPD1 and BnPPD2) and BH478656 and BH720243 from Brassica oleracea (GI: 17686760 and GI: 18820838, BoPPD1 and BoPPD2), were found by BLASTn. The multiple alignments of the deduced PPD sequences and its homologs using ClustalW (Higgins et al., 1996
Subsequently, homologs of RsPPD were searched against the C. reinhardtii expressed sequence tag (EST) index of the Kazusa DNA Research Institute because C. reinhardtii has an enzyme that catalyzes the conversion of Pheid to PyroPheid (Doi et al., 2001 ). However, no homologs were found in the C. reinhardtii EST (http://www.kazusa.or.jp/en/plant/chlamy/EST) or in the CyanoBase (http://www.kazusa.or.jp/cyano/cyano.html). A homology search at the C. reinhardtii genomic database Chlamy Center (http://www.chlamy.org/chlamydb.html) was also done, but no significant homolog was found. This indicates that Pheid demethoxycarbonylase from C. reinhardtii and PPD from radish are genetically different enzymes, which was expected because their reaction mechanisms differ, although they are both involved in the reaction for synthesizing PyroPheid.
In this study, senescence-induced RsPPDs were purified from cotyledons of radish to homogeneity and their amino acid sequences were deduced along with the full cDNA sequence using RT-PCR and RACE. A substrate specificity study showed that these enzymes were extremely specific for Pheids of the dihydroporphyrin and tetrahydroporphyrin types, indicating that they are responsible for the reaction of those structures.
Total RsPPD activity markedly increased with senescence. This phenomenon is predominantly due to up-regulation of proteins by senescence. In this sense, this enzyme, PaO, is one of the senescence-induced genes as well as a porphyrin-opening enzyme in the Chl degradation pathway (Matile et al., 1999
The molecular mass of non-heat-treated, denatured samples of RsPPD was estimated to be in the range of 77 to 61.3 kD, depending on the concentration of polyacrylamide gel used (see Fig. 4). Combined with the results of the deduced amino acid (28,000) and gel filtration (113,000), RsPPD is probably a tetramer. Moreover, the peptide, with a molecular mass of 77 to 61.3 kD, seems to maintain its dimeric form after being denatured, but not in non-heat-treated conditions, although it had a somewhat large molecular mass. This suggests RsPPD is a strongly associated dimer. After heat treatment of RsPPD, three bands, 16.8, 15.9, and 11.8 kD, appeared on SDS-PAGE, but the 16.8- and 15.9-kD peptides overlapped some of the amino acid sequences. Excluding the overlapped peptide, the sum of the molecular mass of the other bands was 28.6 kD. The sizes of the deduced peptide fragments, N-terminal and C-terminal fragments, from the cleavage site were 12,800 and 15,100, respectively, corresponding to the approximate band sizes. As shown in Figure 6, RsPPD was cleaved at a nonconserved amino acid site (gap structure). Based on the strong dimer formation and the fragile primary structure, it is likely that RsPPD forms a dimer by three-dimensional domain swapping (Jaskólski, 2001
In the degradation pathway of Chl, Pheid is catabolized by PaO via red Chl catabolites to NCC and further modified (Matile et al., 1999
Several NCC derivatives were found in different plant species (Matile et al., 1999
It is considered that Chl degradation proceeds only in thylakoids and inner envelope membranes (Matile et al., 1999
Plant Materials
Cotyledons of radish (Raphanus sativus) were purchased from a local market. Induction of senescence was performed according to the method described previously (Suzuki and Shioi, 1999
Chl a, Pheid a, and PyroPheid a were purchased from Wako Pure Chemical Industries. The concentrations of Chls and their derivatives were determined spectrophotometrically as described previously (Shioi et al., 1991
Chl a/b was extracted from the leaves of spinach (Spinacia oleracea) with acetone and partially purified by precipitation with dioxane (Iriyama et al., 1974
Chlid species were prepared from pure respective Chls by the action of chlorophyllase, which catalyzes the hydrolysis of esterified alcohols. Chlorophyllase was obtained from mature leaves of Chenopodium album as described previously (Tsuchiya et al., 1997
The activity of PPD was assayed basically according to the methods described by Shioi et al. (1996b)
Senescent cotyledons of radish (approximately 5,000 g) were homogenized with 20 mM phosphate buffer (pH 7.0) at 4°C in a blender. The homogenate was filtered through six layers of cotton gauze and centrifuged at 17,000g for 30 min at 4°C. The cell-free extract was incubated at 60°C for 10 min and centrifuged at 12,000g for 15 min. The supernatant was fractionated with 60% acetone. The precipitate was dissolved in a small volume of 20 mM Tris-HCl (pH 7.5). After clarifying the solution by centrifugation at 12,000g for 15 min, the enzyme solution was applied to a column of DEAE-Toyopearl 650 M (2.5 x 8 cm; Tosoh) previously equilibrated with 20 mM Tris-HCl (pH 7.5) and eluted with the same buffer containing a linear gradient of NaCl (0–0.35 M). Fractions eluted at 0.1 to 0.14 M NaCl were classified as type 1 and at 0.15 to 0.2 M as type 2 and were pooled separately (see Fig. 3). The following purification procedures were carried out separately for types 1 and 2, but the procedures used for both types 1 and 2 are described together. After adding ammonium sulfate to 30% saturation, the enzyme solution was applied to a butyl-Toyopearl 650 M column (2.5 x 8 cm; Tosoh) previously equilibrated with 20 mM Tris-HCl (pH 7.5) containing 30% saturation of ammonium sulfate and eluted with the same buffer containing a reverse-linear gradient of ammonium sulfate (30%–0% saturation). Fractions eluted at about 0% to 6% saturation for type 1 and 7% to 12% saturation for type 2 were pooled. The active fraction was dialyzed against 20 mM Tris-HCl (pH 7.2) and charged onto a column of Mono-Q (Amersham Biosciences) that had been equilibrated with the same buffer using the ÄKTAFPLC system (Amersham Biosciences). The column was eluted with the same buffer containing a linear gradient of NaCl (0–0.45 M). Fractions eluted at 0.22 M NaCl for type 1 and 0.27 M NaCl for type 2 were pooled. The pooled enzyme solution was concentrated by Centriflo CF25 Membrane Cones (Millipore) and applied to a column of Superdex 200 (Amersham Biosciences) equilibrated with 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl and eluted with the same buffer. Active fractions were collected and used for the subsequent experiments.
Cj-NCC prepared from Cercidiphyllum japonicum was kindly provided by Professor Bernhard Kräutler of Leopold-Franzens-Universität, Innsbruck, Austria. HPLC was carried out with a model LC-10AT (Shimadzu) equipped with a column-temperature controller, using 5-µm Hypersil octadecyl silica (250 x 4.6 mm; Agilent Technologies). Analysis of NCC was performed according to the method of Oberhuber et al. (2001)
Purified PPD was electrophoresed on a 15% SDS-polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane. The protein bands visualized with Coomassie Brilliant Blue were cut out and subjected to amino acid sequencing on a protein sequencer PPSQ-21A (Shimadzu) according to the procedure provided by the manufacturer.
Total RNA was isolated from 1-d-senescent cotyledons of radish by the guanidinium thiocyanate method (Chomczynski and Sacchi, 1987
The cloning of the PPD gene (PPD) was achieved in several steps. The first step was performed on template cDNA synthesis using ReverTra Ace- From a BLAST search using the results of RT-RCR and 3'-RACE, the Arabidopsis (Arabidopsis thaliana) homolog At4g16690, which shows 84% identity, was found. Full-length cDNA was amplified using primers containing the following restriction enzyme site: a forward primer based on a homolog sequence, 5'-TCCCCGGGAATGGGAGGAGAAGGTGGTGCTGA-3', and a reverse primer based on the specific sequence 5'-ATGAATTCTTATAGTTGAAGAAAAGATACAGCAC-3'. PCR was performed using TaKaRa Taq and the template cDNA with primers for 40 cycles at 97°C for 0.5 min and 45°C and 72°C for 1 min each. All PCR products were cloned into pT7-Blue T-Vector (Novagen) and sequenced.
The analogous genomic and amino acidic sequences of PPD were obtained by searching against nonredundant NCBI databases (http://www.ncbi.nlm.nih.gov/BLAST), GenomeNet BLAST2 (http://blast.genome.jp), and TAIR BLAST 2.0 (http://www. arabidopsis.org/Blast). Homology searches at the Chlamydomonas reinhardtii EST index of the Kazusa DNA Research Institute (http://www.kazusa.or.jp/en/plant/chlamy/EST), the C. reinhardtii genomic database, Chlamy Center (http://www.chlamy.org/chlamydb.html), and the CyanoBase (http://www.kazusa.or.jp/cyano/cyano.html) were also done.
The PPD coding region was amplified by RT-PCR using total RNA isolated from 1-d-senescent cotyledons of radish as a template. The 5' primer was 5'-TCCCCGGGAATGGGAGGAGAAGGTGGTGCTGA-3' and the 3' primer was 5'-ATGAATTCTTATAGTTGAAGAAAAGATACAGCAC-3'. The PPD fragment was digested with SmaI and EcoRI restriction enzymes (Toyobo) and cloned into the SmaI and EcoRI sites of the expression vector pGEX-2T. The plasmid pGEX-2T-PPD was used to transform the Escherichia coli BL21(DE3)pLysS strain. Expression of recombinant PPD protein was induced by addition of isopropyl The GST fusion protein was purified using GSTrap FF (1 mL; Amersham Biosciences) and a template program for GSTrap of an ÄKTAprime (Amersham Biosciences) liquid chromatography system according to the manufacturer's protocol. To yield the native protein from GST-PPD, the fusion protein was digested with thrombin. The purified PPD was used as an antigen to raise polyclonal antisera in guinea pigs. Polyclonal antibody was prepared by standard protocol and used without further purification.
SDS-PAGE was performed by the method of Laemmli (1970)
Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce) with bovine serum albumin as a standard. For column chromatographic fractions, protein was assayed spectrophotometrically by measuring at 280 nm at room temperature. Sequence data from this article can be found in the GenBank/EMBL/DDBJ data libraries under accession number AB218276.
We thank Professor Bernhard Kräutler (Leopold-Franzens-Universität, Innsbruck, Austria) for providing the Cj-NCC used in this study. We also thank Keiko Fukuda, Keiko Soga, and Keiko Furuya for technical assistance. Received September 15, 2005; returned for revision November 10, 2005; accepted December 6, 2005.
1 This work was supported by the Ministry of Education, Science, Sports and Culture of Japan (grant nos. 12640631 and 07856). 
2 Present address: National Institute for Longevity Sciences, Obu, Aichi 474–8522, Japan. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yuzo Shioi (sbysioi{at}ipc.shizuoka.ac.jp). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.071290. * Corresponding author; e-mail sbysioi{at}ipc.shizuoka.ac.jp; fax 81–54–238–0986.
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Plant J 21: 189–198[CrossRef][Web of Science][Medline] Ziegler R, Blaheta A, Guha N, Schönegge B (1988) Enzymatic formation of pheophorbide and pyropheophorbide during chlorophyll degradation in a mutant of Chlorella fusca Shihira et Kraus. J Plant Physiol 132: 327–332 This article has been cited by other articles:
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ABSTRACT
RESULTS
, Chls; 
, PPD activity.
, Protein concentrations were estimated with A280; 
(Toyobo) according to the manufacturer's instructions. Total RNA from 1-d-senescent radish leaves was used as a template with oligo(dT) primer to produce single-strand cDNA. For the next PCR, which was RT-PCR (Frohman et al., 1988
-D-thiogalactopyranoside and cultivation for 2 h. Bacteria were collected by centrifugation at 5,500g for 5 min. 
