Linked Life Data

18846292

Source:http://linkedlifedata.com/resource/pubmed/id/18846292

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
2008-10-10
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1474-905X
pubmed:author
pubmed:issnType
Print
pubmed:volume
7
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1260-6
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme.
pubmed:affiliation
Department of Biological Science, Faculty of Science, Shizuoka University, 422-8529, Shizuoka, Japan.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't