Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme.
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.) 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(-1) 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 Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs.
Suzuki
Yasuyo
Y
Department of Biological Science, Faculty of Science, Shizuoka University, 422-8529, Shizuoka, Japan.
Soga
Keiko
K
Yoshimatsu
Katsuhiko
K
Shioi
Yuzo
Y
D055162:Biocatalysis; D015153:Blotting, Western; D002734:Chlorophyll / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D031224:Raphanus / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; C040298:pyropheophorbide a; D005982:Glutathione Transferase; D004950:Esterases