Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 289, Issue 34, Pages 23859-23869Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.571703
Keywords
Enzyme Mechanism; Enzyme Mutation; Plant; Plant Biochemistry; Plant Defense; Terpenoid; Terpenoid Synthase
Categories
Funding
- Natural Sciences and Engineering Research Council of Canada
- Genome Canada
- Genome BC
- Genome Quebec
- Genome Science and Technology program
- British Columbia Epilepsy Society
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Background: (+)-3-Carene/(-)-sabinene synthases are associated with insect resistance in Sitka spruce. Results: Amino acid position 596 is critical for the monoterpene synthase product profile. Conclusion: This work revealed mechanistic underpinnings for patterns of functional evolution of this monoterpene synthase family. Significance: Catalytic plasticity of conifer monoterpene synthases is a major factor in the diversification of the TPS-d family and evolution of insect resistance. 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 multiproduct (+)-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 (+)-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, whereas phenylalanine promotes (-)-sabinene. Homology modeling predicts that 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 defense against insects.
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