Cytochrome P450-Catalyzed Biosynthesis of a Dihydrofuran Neoclerodane in Magic Mint (Salvia divinorum)

Abstract

The hallucinogenic plant, Salvia divinorum, synthesizes neoclerodane diterpenes, such as salvinorins, salvidivins, and salvinicins, which are agonistic or antagonistic to μor κ-opioid receptors. From S. divinorum trichomes, crotonolide G synthase (SdCS; CYP76AH39) was identified. It catalyzes the conversion of kolavenol to a dihydrofuran neoclerodane, crotonolide G. 18 O 2feeding studies confirmed that SdCS incorporates an aerobic oxygen into crotonolide G, rather than forming a cation at C16 that is trapped by the alcohol at C15. Structural modeling of SdCS accompanied by site-directed mutagenesis established the importance of V367 and F479 residues in substrate-binding. The dihydrofuran neoclerodane can serve as a unique lead structure for drug development.

The genomes of medicinal skullcaps reveal the polyphyletic origins of clerodane diterpene biosynthesis in the family Lamiaceae

“…Although in S. splendens the dedicated KLS SspdiTPS1.5 is placed in the clade with other class I diTPS that act on CPP, it is phylogenetically distant (Figure 5A) from the promiscuous miltiradiene synthase SspdiTPS1.3 which also shows KLS enzymatic activity, and from the KLS/IKLS enzymes from Scutellaria species. The paired activity of class II and class I diTPS in plastids, is essential for the generation of kolavenol and later utilization by downstream enzymes (De La Peña and Sattely, 2021; Kwon et al ., 2022). The phylogeny of class I clerodane synthases supports the polyphyletic origin of clerodane biosynthetic pathway in the family Lamiaceae.…”

“…The copyright holder for this preprint this version posted September 26, 2022. ; https://doi.org/10.1101/2022.09.25.509435 doi: bioRxiv preprint Scutellaria species. The paired activity of class II and class I diTPS in plastids, is essential for the generation of kolavenol and later utilization by downstream enzymes (De La Peña and Sattely, 2021; Kwon et al, 2022). The phylogeny of class I clerodane synthases supports the polyphyletic origin of clerodane biosynthetic pathway in the family Lamiaceae.…”

“…kolavenol and isokolavenol, respectively (Figure 3). These class I diTPSs catalyse the synthesis of either kolavenol or isokolavenol and represent activities involved in the second step in the clerodane biosynthetic pathway (Kwon et al, 2022;Muchlinski et al, 2021). Heterologously expressed (E. coli) and purified SbdiTPS1.3, a class I diTPS from S. baicalensis, catalysed the transformation of IKPP to isokolavenol in coupled enzyme assays with SbdiTPS2.7 and SbdiTPS2.8 (Figure 3).…”

“…The proposed mechanism for class II clerodane synthases begins with the initial protonation and cyclization of GGPP for the formation of the intermediate ent -labda-13 E -en-8-yl diphosphate carbocation; a Wagner-Meerwein rearrangement takes place with two subsequent 1, 2 hydride and methyl shifts resulting to the generation of the clerodane scaffold with the formation of kolav-13E-en-4-yl diphosphate carbocation, and a final proton abstraction results in the formation of either KPP or IKPP (Figure S1) (Jia et al ., 2019; Jia et al ., 2016; Lemke et al, 2019; Potter et al, 2016). No class I diTPSs have yet been described which act on KPP or IKPP in a native pathway (Chen et al ., 2017; Kwon et al, 2022; Pelot et al ., 2017), limiting the elucidation of clerodane diterpenoid biosynthesis in the mint family.…”

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“…Crotonolide G synthase (SdCS), a cytochrome P450 enzyme from Salvia divinorum , has been shown to catalyse the formation of the dihydrofuran of crotonolide G 29 from kolavenol 28 (Scheme 1) during the biosynthesis of diterpenes such as salvinorins, salvidivins and slavinicins. 21 Feeding studies using 18 O 2 , revealed that SdCS facilitates incorporation of an aerobic oxygen atom at C-16 of 28 , with dihydrofuran formation then achieved by nucleophilic attack of the resulting alcohol on the activated C-15 hydroxyl group. Genome mining and activation of a cryptic polyketide synthase gene from Aspergillus hancockii has allowed the discovery of hancockinone A 30 , a prenylated polycyclic aromatic polyketide with a 6/6/6/5 skeleton.…”