Improved selectivity of an engineered multi-product terpene synthase

Abstract

Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a k_cat/K_M of 0.62 mM⁻¹ s⁻¹, which is nearly identical to that of the parent Cop2, which had a k_cat/K_M of 0.58 mM⁻¹ s⁻¹.