Directed Evolution Experiments Reveal Mutations at Cycloartenol Synthase Residue His477 that Dramatically Alter Catalysis

Cycloartenol synthase cyclizes and rearranges oxidosqualene to the protosteryl cation and then specifically deprotonates from C-19. To identify mutants that deprotonate differently, randomly generated mutant cycloartenol synthases were selected in a yeast lanosterol synthase mutant. A novel His477Asn mutant was uncovered that produces 88% lanosterol and 12% parkeol. The His477Gln mutant produces 73% parkeol, 22% lanosterol, and 5% Δ7-lanosterol. These are the most accurate lanosterol synthase and parkeol synthase that have been generated by mutagenesis.