Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase.  ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two-carbon unit derived from malonyl-CoA.  This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum, as a model for the family of ELOs.  EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1?9 to 18:1?11.  To examine their potential role in the condensation reaction, site-directed mutagenesis was used to change residues highly conserved among the ELO family.  Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry.  Sixteen out of twenty mutants had a decrease in 18:1?11 production when compared to wild-type EloA.  Ten out of these sixteen mutants showed little to no activity while the remainder had 10-60% of wild-type activity.  Immunoblot analysis using antiserum raised against a synthesized EloA peptide indicated that the protein levels of most of the mutant EloAs with little to no activity were comparable to that of the wild-type suggesting that the decrease in activity was not the result of decreased expression.   For example, mutant K123A showed little activity but no decrease in protein expression suggesting that this residue plays a role in substrate-binding or catalysis.  Progress toward the isolation of hexahistidine-EloA fusion proteins will be reported

Characterization of fatty acid elongase condensing enzyme by site-directed mutagenesis and biochemical analysis

246th National Meeting (2013)

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