This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. <i>Oleate desaturases</i> belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from <i>Crepis alpina</i>, and a number of point mutants have been expressed in <i>Saccharomyces cerevisiae</i>, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis.

This study provides insight into the structure/function relationship between desaturases and acetylenases, and indicates amino acid residues within acetylenases which influence reaction outcome. <i>Oleate desaturases</i> belong to a family of enzymes capable of introducing cis double bonds between C12 - C13 in oleate esters. Acetylenases are a subset of oleate desaturase enzymes which introduce a triple bond in the C12 - C13 position of linoleate. To better understand which amino acids could be responsible for differentiating the activity of acetylenases from typical desaturases, a total of 50 protein sequences were used to compare the two classes of enzymes resulting in the identification of 11 amino acid residues which are conserved within either separate family but differ between the two groups of enzymes. These identified amino acid residues were then singularly altered by site-directed mutagenesis to test their role in fatty acid modification. Specifically, the wild type acetylenase, Crep1 from <i>Crepis alpina</i>, and a number of point mutants have been expressed in <i>Saccharomyces cerevisiae</i>, followed by fatty acid analysis of the resulting cultures. Results indicate the importance of 4 amino acid residues within Crep1 (Y150, F259, H266, and V304) with regards to desaturase and acetylenase chemoselectivity, stereoselectivity, and/or substrate recognition. The F259L mutation affected the acetylenase by converting it to an atypical FAD2 capable of producing both cis and trans isomers. The V304I mutation resulted in the conversion of Crep1 into a stereoselective FAD2, where only the cis isomers of 16:2 and 18:2 were produced. The Y150F mutation led to a loss of acetylenase activity without affecting the inherent desaturase activity of Crep1. The H266Q mutation appears to affect substrate selection causing an inability to bind substrate (16:1-9c and/or 18:1-9c) in a cisoid conformation, resulting in an increased accumulation of trans product. The changes in enzyme activity detected in cultures expressing Crep1 mutants demonstrate the profound effect that exchanging as little as one amino acid can have on an enzyme properties. Enzymes retain some conservation of amino acids necessary for activity, such as those involved in metal ion binding, whereas subtle changes can affect overall enzyme function and catalysis.