Site-Directed Mutagenesis Reveals Critical Amino Acids and an Enzymatic Mechanism for Product Profiles of (+)-3-Carene Synthases and Sabinene Synthase in Conifer Defence

The White Pine Weevil is one of the most devastating pests of Sitka spruce trees. Variation in the levels of the monoterpene (+)-3-carene has been associated with resistance to weevil attack. This variation in (+)-3-carene biosynthesis is due to variations of gene copy number, transcript and protein levels, and catalytic efficiency in a small family of (+)-3-carene synthases-like genes. This family includes the three (+)-3-carene synthases PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, as well as the sabinene synthase PsTPS-sab. PsTPS-3car2 is responsible for the relatively high levels of (+)-3-carene in weevil-resistant Sitka spruce clones. Due to their high sequence similarity yet different product profiles, comparisons of PsTPS-sab and PsTPS-3car2 were used to guide site-directed mutagenesis and domain swapping to explore the amino acids responsible for their specific product profiles. More than ~66% of the PsTPS-3car2 specific product profile was found to be due to only three amino acids in the helix J region; G595, L596, and F599 with L596 alone being responsible for ~50% of the product profile. Similarly, 100% of PsTPS-sab’s product profile was found to be due to the amino acids in the same three positions with F596 alone being responsible for ~85% of the product profile. Homology modeling predicts the amino acid in the 596 position to sterically affect the carbocation position in the reaction intermediate, directly affecting the specific product profile. Investigation of the amino acids responsible for both product profile and kinetic parameters of PsTPS-3car2 provides insights into the evolution of this small family of (+)-3-carene synthase-like monoterpene synthases associated with insect resistance in Sitka spruce.