The induced evolution of Arabidopsis thaliana cycloartenol synthase and the cloning of Oryza sativa cycloartenol synthase
Darr, Lisa Buckingham
Master of Arts
In the biosynthesis of cholesterol in mammals and ergosterol in fungi, oxidosqualene is cyclized to lanosterol by lanosterol synthase. Similarly, the biosynthesis of plant sterols includes the conversion of the same substrate, oxidosqualene, to cycloartenol by cycloartenol synthase. Cloned lanosterol synthases and cycloartenol synthases are about 30% similar, have over 100 conserved residues and have structurally similar cyclization products. I explored the dissimilarities between these two cyclases by inducing the evolution of a yeast lanosterol synthase mutant expressing cycloartenol synthase. Cycloartenol synthase mutants with lanosterol synthase activity were identified by selecting transformants that were no longer sterol auxotrophs. Once I identified the site of the mutation, I partially characterized the cyclization products of two cycloartenol synthase mutants with lanosterol synthase activity. I also cloned a large fragment of the Oryza sativa cycloartenol synthase gene, as concluded by the deduced amino acid sequence.