title: The gymnosperm cytochrome P450 CYP750B1 catalyzes stereospecific monoterpene hydroxylation of (+)-sabinene in thujone biosynthesis in western redcedar.
abstract: Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and β-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: Andreas
authors.identifier: http://orcid.org/0000-0002-9614-7079
authors.initials: A
authors.lastname: Gesell
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: Markus
authors.initials: M
authors.lastname: Blaukopf
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: Lina
authors.identifier: http://orcid.org/0000-0003-4161-2540
authors.initials: L
authors.lastname: Madilao
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: Macaire M S
authors.initials: MM
authors.lastname: Yuen
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: Jim
authors.initials: J
authors.lastname: Mattsson
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.).
authors.forename: John H
authors.initials: JH
authors.lastname: Russell
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 (A.G., L.M., M.M.S.Y., J.B.);Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1 (M.B., S.G.W.);Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 (J.M.); andBritish Columbia Ministry of Forests, Lands and Natural Resource Operations, Cowichan Lake Research Station, Mesachie Lake, British Columbia, Canada V0R 2N0 (J.H.R.) bohlmann@msl.ubc.ca.
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
mesh_terms: D000081008:Bicyclic Monoterpenes; D055162:Biocatalysis; D053898:Biosynthetic Pathways; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D056726:Genetic Association Studies; D006900:Hydroxylation; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D010671:Phenylurea Compounds / Q000378:metabolism; D010802:Phylogeny; D024301:Plant Bark / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism; D029783:Thuja / Q000201:enzymology* / Q000235:genetics; D014020:Tissue Extracts
chemical_list: D000081008:Bicyclic Monoterpenes; D039821:Monoterpenes; D010671:Phenylurea Compounds; D012333:RNA, Messenger; D013729:Terpenes; D014020:Tissue Extracts; D009243:NAD; C052984:sabinol; C028904:isoproturon; C035127:sabinene; C005790:beta-thujone; D003577:Cytochrome P-450 Enzyme System
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