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.

Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures

“…It has been proposed previously that the twostep cyclization of the universal diterpenoid precursor geranylgeranyl diphosphate, via (+)-copalyl diphosphate, yields miltiradiene as an abietane hydrocarbon intermediate , and our data agree with this suggestion. Oxidation at C18, like that described for CYP720 in conifers (Ro et al, 2005;Hamberger et al, 2011;Geisler et al, 2016), would produce dehydroabietic acid (which we detected in T. regelii root cultures; Fig. 2).…”

Cell‐type‐ and tissue‐specific transcriptomes of the white spruce (Picea glauca) bark unmask fine‐scale spatial patterns of constitutive and induced conifer defense

“…Expression of several different P450 genes was also highly CRD specific (Figure ). The two most highly expressed P450s in the CRD cells, and among the most highly expressed P450s in all cell types, were CYP720B4 and CYP720B12, which are known to function in diterpene resin acid biosynthesis (Hamberger et al ., ; Geisler et al ., ; Figure ).…”

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms

“…Further studies are required to verify empirically whether this reaction occurs spontaneously or is catalyzed by a yet unidentified enzyme. Given the typically oxygenated nature of known diterpenoids in T. cryptomerioides and other gymnosperms (Ro et al ., ; Hamberger et al ., ; Geisler et al ., ), it is plausible that the observed Tc diTPS products do not represent pathway end products in planta , but are further modified by P450s and/or other enzyme classes.…”