Polyenoic fatty acid isomerase from the marine alga Ptilota filicina: protein characterization and functional expression of the cloned cDNA.
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.
Zheng
Wei
W
Calgene Campus of Monsanto Company, 1920 Fifth Street, Davis, California 95616, USA. wei.zheng@monsanto.com
Wise
Mitchell L
ML
Wyrick
Annette
A
Metz
James G
JG
Yuan
Ling
L
Gerwick
William H
WH
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015342:DNA Probes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000461:Rhodophyta / Q000201:enzymology* / Q000235:genetics
D015342:DNA Probes; D018076:DNA, Complementary; D011994:Recombinant Proteins; D019748:Carbon-Carbon Double Bond Isomerases; C105113:polyenoic fatty acid isomerase