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

By Selene Hernandez-Buquer

Abstract

Indiana University-Purdue University Indianapolis (IUPUI)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 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. 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 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work

Topics: ELONGASE, CONDENSING ENZYME, Fatty acids -- Synthesis -- Research -- Analysis, Lipids -- Metabolism -- Research, Enzymes -- Analysis, Biochemistry -- Technique, Mutagenesis, Dictyostelium discoideum, Proteins -- Structure, Saccharomyces cerevisiae, Oligonucleotides, Microsomes, Yeast fungi, Biosynthesis -- Research, Gas chromatography, Mass spectrometry, Polymerase chain reaction
Year: 2014
OAI identifier: oai:scholarworks.iupui.edu:1805/5915
Provided by: IUPUIScholarWorks

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