Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: Protein characterization and functional expression of the cloned cDNA

W. Zheng, M. L. Wise, A. Wyrick, J. G. Metz, L. Yuan, W. H. Gerwick

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

The recently described enzyme, polyenoic fatty acid isomerase (PFI), from the marine alga Ptilota filicina J. Argardh has been analyzed with respect to its protein structure and an associated cofactor. The enzyme was purified to homogeneity (as judged by SDS-PAGE and silver staining). By sedimentation equilibrium ultracentrifugation the mass of the native enzyme was estimated to be 125 kDa. The N-terminal peptide sequence derived from this protein was used to isolate two very similar cDNA clones encoding novel 500-amino acid proteins, both with calculated molecular masses of 55.9 kDa and pis of 4.87. The data predict translation of a preprotein containing a signal peptide of 21 amino acids that is removed during maturation. Deglycosylation assays demonstrate that native PFI from P. filicina is a glycoprotein. The purified protein is chromophoric with a flavin-like UV spectrum and sequence analysis reveals the presence of a flavin-binding motif near the mature N-terminus. Heterologous expression of active PFI in Arabidopsis, using one of the cDNA clones, was successful as evidenced by conversion of arachidonic acid to a conjugated triene in an in vitro assay of the transgenic plant tissues.

Original languageEnglish
Pages (from-to)11-20
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume401
Issue number1
DOIs
StatePublished - May 1 2002

Bibliographical note

Funding Information:
A portion of this work was supported by the Oregon Sea Grant Program R/BT-1.

Funding

A portion of this work was supported by the Oregon Sea Grant Program R/BT-1.

FundersFunder number
Oregon Sea Grant ProgramR/BT-1

    Keywords

    • Conjugase
    • Conjugated fatty acids
    • Marine natural products

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology

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