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J Biol Chem. 2014 Aug 22;289(34):23859-69. doi: 10.1074/jbc.M114.571703. Epub 2014 Jul 11.

Plasticity and evolution of (+)-3-carene synthase and (-)-sabinene synthase functions of a sitka spruce monoterpene synthase gene family associated with weevil resistance.

Author information

1
From the Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4 and the Genome Science and Technology Program, University of British Columbia, Vancouver, British Columbia, V5Z 4S6, Canada.
2
From the Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4 and.
3
From the Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4 and the Genome Science and Technology Program, University of British Columbia, Vancouver, British Columbia, V5Z 4S6, Canada bohlmann@msl.ubc.ca.

Abstract

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.

KEYWORDS:

Enzyme Mechanism; Enzyme Mutation; Plant; Plant Biochemistry; Plant Defense; Terpenoid; Terpenoid Synthase

PMID:
25016016
PMCID:
PMC4156071
DOI:
10.1074/jbc.M114.571703
[Indexed for MEDLINE]
Free PMC Article
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