A FAD2 homologue from Lesquerella lindheimeri has predominantly fatty acid hydroxylase activity

A genomic sequence encoding a polypeptide with 91 % sequence identity to the Lesquerella fendleri bifunctional oleate 12-hydroxylase:desaturase was amplified by PCR from Lesquerella lindheimeri. Expression of the gene in the yeast Saccharomyces cerevisiae resulted in the synthesis of ricinoleic acid and very low levels of di-unsaturated fatty acids. Comparison of the amino acid sequences of the L. lindheimeri and castor bean oleate 12-hydroxylase enzymes to those of the L. fendleri bifunctional oleate 12-hydroxylase:desaturase and oleate 12-desaturase enzymes from 32 diverse species identified a single amino acid (M295) that was conserved in the hydroxylases and different but also conserved in the desaturases and the bifunctional enzyme. Site-directed mutagenesis indicated that this residue was most likely not involved in determining the catalytic outcome of the hydroxylation/desaturation reaction. Transformation of an Arabidopsisfad2/fae 1 mutant line with the L. lindheimeri hydroxylase gave further evidence that this enzyme, like the castor oleate 12-hydroxylase, is primarily a fatty acid hydroxylase and should not be considered bifunctional. Total hydroxy fatty acid content of up to 18% of seed fatty acids was measured in homozygous transformants. Lines with the highest hydroxy fatty acid content showed significant reduction in total oil content. Crown Copyright (c) 2007 Published by Elsevier Ireland Ltd. All rights reserved.