Plasticity and Evolution of (+)-3-Carene Synthase and (-)-Sabinene Synthase Functions of a Sitka Spruce Monoterpene Synthase Gene Family associated with Weevil Resistance [Enzymology]

July 11th, 2014 by Roach, C. R., Hall, D. E., Zerbe, P., Bohlmann, J.

The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multi-product (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (−)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3 and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by either (+)-3-carene in PsTPS-3car1, PsTPS-3car2 and PsTPS-3car3 or (−)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene while phenylalanine promotes (−)-sabinene. Homology modeling predicts position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defence against insects.
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