Skip to main content
. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Science. 2023 Jan 26;379(6630):361–368. doi: 10.1126/science.adf1017

Fig. 3. Characterization of melianol oxide isomerases (MOIs).

Fig. 3.

(A) Characterization of products generated via overexpression of MOIs and SI using transient gene expression in N. benthamiana. Liquid chromatography–mass spectrometry (LC-MS) extracted ion chromatograms (EICs) resulting from overexpression of AtHMGR, CsOSC1, CsCYP71CD1, CsCYP71BQ4, CsCYP88A51, and CsMOIs and CsSI in N. benthamiana. Representative EICs are shown (n=3).

(B) Phylogenetic tree (Bayesian) of sterol isomerase (SI) genes from high-quality plant genomes. SI sequences from 33 plant species were identified and downloaded from Phytozome via pFAM assignments (PF05241). Branch supports are provided (excluding those >0.95) and monocot SIs have been used as an outgroup. Enzymes that have melianol oxide isomerase activity when tested by Agrobacterium-mediated expression in N. benthamiana with melianol (1) biosynthetic genes and CsCYP88A51 or MaCYP88A108, have been renamed MOI, e.g. CsMOI1-3 and MaMOI2. Characterized MOIs from C. sinensis and M. azedarach selected for further analysis are bolded and their respective tree branches are indicated in orange. Genes from Citrus are shown in blue and those from Melia are shown in green.

(C) Percentage protein identity of MOIs and SIs from C. sinensis and M. azedarach, those with sequence similarity greater than 75% are highlighted in gray.

(D) Co-expression of MOIs and SIs from C. sinensis and M. azedarach displaying rank and PCC as outlined in Fig. 2A, 2C.

(E) Proposed mechanism of CsCYP88A51/MaCYP88A108, CsMOI2/MaMOI2 and CsMOI1. CsCYP88A51/MaCYP88A108 first oxidizes the C7,C8 position of melianol (1) to yield an unstable epoxide intermediate (2), which can undergo spontaneous C-30 methyl shift from C-14 to C-8 (highlighted in red). Either (2) or the methyl shifted product spontaneously form a series of oxidized products (2a - 2d). In the presence of MOIs, the rearrangement of (2) is guided to form either (3) or (4) and no (2a), (2b), (2c), and (2d) are observed. Structures of (2a), (2b), (2c) and (2d) are not determined but their MS fragmentation patterns suggest they are isomeric molecules resulting from a single oxidation of melianol (1), which doesn’t exclude the possibility them of being (2), (3), or (4) (as shown for Ailanthus altissima CYP71BQ17 (35)).