title: Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.
abstract: Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
authors.forename: Katrin
authors.identifier: 0000-0002-7630-1868
authors.initials: K
authors.lastname: Geisler
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
authors.forename: Niels Berg
authors.identifier: 0000-0002-6245-5057
authors.initials: NB
authors.lastname: Jensen
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
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.
authors.forename: Lina
authors.identifier: 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 bohlmann@msl.ubc.ca.
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
mesh_terms: D045784:Abietanes; D000595:Amino Acid Sequence; D001483:Base Sequence; D002264:Carboxylic Acids; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary; D018744:DNA, Plant; D004224:Diterpenes / Q000378:metabolism*; D045786:Diterpenes, Kaurane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D005875:Gibberellins / Q000096:biosynthesis; D008861:Microsomes; D010616:Phenanthrenes; D010802:Phylogeny; D028222:Picea / Q000201:enzymology* / Q000235:genetics; D028223:Pinus / Q000201:enzymology* / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012116:Resins, Plant / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D059467:Transcriptome
chemical_list: D045784:Abietanes; D002264:Carboxylic Acids; D018076:DNA, Complementary; D018744:DNA, Plant; D004224:Diterpenes; D045786:Diterpenes, Kaurane; D005875:Gibberellins; D010616:Phenanthrenes; D010940:Plant Proteins; D012116:Resins, Plant; C115138:isopimaric acid; C002627:kaurene; C051436:kaurenoic acid; C008911:pimaric acid; D003577:Cytochrome P-450 Enzyme System; C082072:sandaracopimaric acid; C023710:abietic acid