Evolution of conifer diterpene synthases: diterpene resin acid biosynthesis in lodgepole pine and jack pine involves monofunctional and bifunctional diterpene synthases.
Diterpene resin acids (DRAs) are major components of pine (Pinus spp.) oleoresin. They play critical roles in conifer defense against insects and pathogens and as a renewable resource for industrial bioproducts. The core structures of DRAs are formed in secondary (i.e. specialized) metabolism via cycloisomerization of geranylgeranyl diphosphate (GGPP) by diterpene synthases (diTPSs). Previously described gymnosperm diTPSs of DRA biosynthesis are bifunctional enzymes that catalyze the initial bicyclization of GGPP followed by rearrangement of a (+)-copalyl diphosphate intermediate at two discrete class II and class I active sites. In contrast, similar diterpenes of gibberellin primary (i.e. general) metabolism are produced by the consecutive activity of two monofunctional class II and class I diTPSs. Using high-throughput transcriptome sequencing, we discovered 11 diTPS from jack pine (Pinus banksiana) and lodgepole pine (Pinus contorta). Three of these were orthologous to known conifer bifunctional levopimaradiene/abietadiene synthases. Surprisingly, two sets of orthologous PbdiTPSs and PcdiTPSs were monofunctional class I enzymes that lacked functional class II active sites and converted (+)-copalyl diphosphate, but not GGPP, into isopimaradiene and pimaradiene as major products. Diterpene profiles and transcriptome sequences of lodgepole pine and jack pine are consistent with roles for these diTPSs in DRA biosynthesis. The monofunctional class I diTPSs of DRA biosynthesis form a new clade within the gymnosperm-specific TPS-d3 subfamily that evolved from bifunctional diTPS rather than monofunctional enzymes (TPS-c and TPS-e) of gibberellin metabolism. Homology modeling suggested alterations in the class I active site that may have contributed to their functional specialization relative to other conifer diTPSs.
Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4.
Dawn E
DE
Hall
Philipp
P
Zerbe
Sharon
S
Jancsik
Alfonso Lara
AL
Quesada
Harpreet
H
Dullat
Lina L
LL
Madilao
Macaire
M
Yuen
Jörg
J
Bohlmann
D019883:Alkyl and Aryl Transferases / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D055162:Biocatalysis; D002264:Carboxylic Acids / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004224:Diterpenes / Q000032:analysis* / Q000378:metabolism; D019143:Evolution, Molecular; D008401:Gas Chromatography-Mass Spectrometry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010616:Phenanthrenes / Q000032:analysis / Q000378:metabolism; D010802:Phylogeny; D028223:Pinus / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D059467:Transcriptome / Q000235:genetics
D002264:Carboxylic Acids; D018076:DNA, Complementary; D004224:Diterpenes; D010616:Phenanthrenes; C115138:isopimaric acid; C008911:pimaric acid; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; C082072:sandaracopimaric acid