Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

• Published in: FEBS letters 2013-11, Vol.587 (23), p.3837-3842
• Main Authors: Hernandez-Buquer, Selene, Blacklock, Brenda J.
• Format: Article
• Language: English
• Subjects: 3-ketoacylCoA synthase; Acetyltransferases - chemistry; Acetyltransferases - genetics; Acetyltransferases - metabolism; Alanine - chemistry; Alanine - genetics; Amino Acid Sequence; Catalytic Domain; CoA; coenzyme A; Condensing enzyme; Dictyostelium - enzymology; Dictyostelium discoideum; ELO; endoplasmic reticulum; Fatty acid elongation; gas chromatography/mass spectrometry; GC/MS; KCS; Molecular Sequence Data; Mutagenesis, Site-Directed; very long chain fatty acids; VLCFA
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2013.10.011

•20 Highly conserved residues in EloA were mutated.•Many mutants were inactive yet were expressed at levels comparable to the wild-type.•Mutation of the only conserved Cys did not abolish activity.•ELOs use a reaction mechanism distinct from that of the Claisen-like enzymes. The condensation step of fatty acid elongation is the addition of a C2 unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.