Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme

Yasuyo Suzuki, Keiko Soga, Katsuhiko Yoshimatsu, Yuzo Shioi
2008 Photochemical and Photobiological Sciences  
Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13 2 -carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.)
more » ... were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg/L of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the K m values toward Pheid a: 95.5 μM for PPD-GST and about 15 μM for native PPDs. The first committed step in the early stage of Chl a degradation is hydrolysis of the phytyl ester linkage catalyzed by chlorophyllase (EC 3.1.1.14), which forms chlorophyllide a and phytol. Molecular cloning of the chlorophyllase gene from Chenopodium album 12 and Citrus fruit 13 was performed recently. At the same time, the coronatine-induced gene ATHCOR1 was determined to be the gene encoding chlorophyllase by homology searches of sequences and expression of its gene from Arabidopsis thaliana. 12 Subsequently, magnesium is released from the macrocyclic ring and Pheid a is formed. Previously, we demonstrated that an activity catalyzing this reaction is not due to a protein, but to a low-molecular-mass, heat-stable substance. 14 The highly purified substance from C. album was specific not only for Mg 2+ , but also for several divalent cations, and therefore we named it "metal-chelating substance". 15 Recent studies surveying the entire Mg-dechelatase activity in extracts from C. album using a native substrate confirmed these facts. 16, 17 The metal-chelating substance is thus a possible candidate for the catalyst of the Mg-dechelating reaction. The formed Pheid is shown to be converted into red Chl catabolite by Pheid a oxygenase and metabolized further into small compounds in the main Chl degradation 4 pathway. 5, 10 In some restricted orders of higher plants and algae, an auxiliary step of macrocyclic ring modification occurs further when the formed Pheid a is converted to pyropheophorbide (PyroPheid) a. [18] [19] [20] Two kinds of enzymes that catalyze alternative reactions in the formation of PyroPheid a were found. One enzyme, called pheophorbidase (PPD), is an esterase that catalyzes demethylation of the methoxycarbonyl group of Pheid a to a precursor of PyroPheid a, identified as C-13 2 -carboxylPyroPheid a; next, non-enzymatic decarboxylation of the precursor occurs, leading to PyroPheid a. The other enzyme, termed Pheid demethoxycarbonylase, was purified from the Chl b-less mutant NL-105 of Chlamydomonas reinhardtii. 21 This enzyme produced no intermediate, unlike the PPD reaction, indicating that Pheid a is converted directly to PyroPheid a, probably by the acetyl transferase reaction. Recently, we purified two types of PPD from senescent cotyledons of radish (Raphunus sativus L.) to homogeneity and determined the biochemical and molecular properties, including cDNA cloning. 22 The enzyme activity was separated into two peaks by chromatography on a DEAE-Toyopearl column. These enzymes, termed type 1 and type 2 according to their order of elution in the chromatography, correspond to senescence-induced and constitutive enzymes, respectively. Two types of PPD were found in radish, but only one PPD gene, named PPD, was obtained in spite of several screenings of radish cDNAs. The deduced polypeptide has a lipase domain that was presumed to be the active site of the esterase, as in the case of chlorophyllase. 12 These enzymes are considered to be involved in the formation of PyroPheid because of their extremely specific substrate specificity for Pheids, and possibly to be responsible for the production of PyroPheid, especially in the anaerobic state, 21 in which the next oxygen-requiring reaction catalyzed by Pheid a oxygenase in the main Chl degradation using gel filtration instead of the affinity column. The tag-free PPD purified by HPLC showed fairly low activity, although it was detectable with a CBB stain. The purified protein was used as an antigen to raise a polyclonal antiserum in guinea pig. This polyclonal antibody recognized PPD-GST, tag-free PPD, and native PPD, indicating that the purified protein is PPD. Comparison between biochemical properties of recombinant PPD-GST and native PPDs The PPD-GST fusion protein, tag-free PPD, and native PPD were assayed by immunoblotting (Fig. 5) . Removal of the GST tag from PPD-GST fusion protein was examined by using anti-PPD antiserum. The soluble fusion protein showed one major band with a molecular mass of 55 kDa and two smaller size bands, one of which, a 44 kDa protein, is probably due to degraded PPD-GST as described above (see Fig. 2B ), and the other is an unknown 33 kDa protein (Fig. 5, lane 1) . The thrombin-treated, tag-free recombinant PPD showed a molecular mass of 31 kDa in addition to the non-digested PPD-GST (55 kDa) and the unknown 33 kDa protein (Fig. 5, lane 2) . The 33 kDa protein emerged only in the expression system using E. coli, and it may be due to non-specific binding of the polyclonal antiserum for PPD to protein derived from E. coli. In the non-heated conditions, the native PPD showed a molecular mass of 77 kDa with a slightly cleaved band (Fig. 5, lane 3) , while PPD-GST had a main activity peak at a molecular mass of 138 kDa and also 309 kDa, but not at 55 kDa after gel filtration (data not shown). The weaker staining of this band is probably due to weak reactivity to antiserum. When the native protein was heat-treated in a sample buffer containing SDS and 2-mercaptoethanol, the band was cleaved into three fragments (16.8, 15.9, and 11.8 kDa). 22 In the present immunoblot analysis, two bands corresponding to
doi:10.1039/b810271f pmid:18846292 fatcat:kaiyivck5jdpnoztzvxyavfi2q