Journal Article

Overexpression, Enzymatic Properties and Tissue Localization of a Ferrochelatase of Cucumber

Takuo Suzuki, Tatsuru Masuda, Hachiro Inokuchi, Hiroshi Shimada, Hiroyuki Ohta and Ken-ichiro Takamiya

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 41, issue 2, pages 192-199
Published in print February 2000 | ISSN: 0032-0781
Published online February 2000 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/41.2.192
Overexpression, Enzymatic Properties and Tissue Localization of a Ferrochelatase of Cucumber

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  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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Ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to generate protoheme. A putative mature region of a cucumber ferrochelatase cDNA (hemH) was overexpressed in Escherichia coli and purified to homogeneity (40 kDa). The optimum pH was 7.7, and the apparent Km values for deuteroporphyrin IX and Fe2+ were 14.4 μM and 4.7 μM, respectively. The activity of the ferrochelatase was inhibited by N-methylprotoporphyrin IX (I50 = 4nM). Western blot analysis with a polyclonal antibody raised against the recombinant ferrochelatase showed that the antibody crossreacted with protein extracts from hypocotyls and roots of cucumber but not with that from cotyledons. The antibody did not crossreact with proteins of thylakoid membranes of chloroplasts in cucumber cotyledons, although the ferrochelatase activity was mainly associated with the thylakoid membranes. Northern blot analysis also indicated that the hemH gene was expressed mainly in hypocotyls and roots, but little in cotyledons, and the level of the hemH transcripts was not light-responsive. These results demonstrated that the cucumber hemH gene encodes a ferrochelatase which presumably functions for heme biosynthesis in non-photosynthetic tissues, such as hypocotyls and roots, and suggested the presence of other types of ferrochelatase in cucumber, one of which is located in thylakoid membranes of chloroplasts.

Keywords: Cucumis sativus; Ferrochelatase (EC 4.99.1.1); Functional expression; Heme synthesis; Immunolocalization

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Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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