Allylic hydroxylation of triterpenoids by a plant cytochrome P450 triggers key chemical transformations that produce a variety of bitter compounds

Abstract

Cucurbitacins are highly oxygenated triterpenoids characteristic of plants in the family Cucurbitaceae and responsible for the bitter taste of these plants. Fruits of bitter melon (Momordica charantia) contain various cucurbitacins possessing an unusual ether bridge between C5 and C19, not observed in other Cucurbitaceae members. Using a combination of next-generation sequencing and RNA-Seq analysis and gene-to-gene co-expression analysis with the ConfeitoGUIplus software, we identified three P450 genes, CYP81AQ19, CYP88L7, and CYP88L8, expected to be involved in cucurbitacin biosynthesis. CYP81AQ19 co-expression with cucurbitadienol synthase in yeast resulted in the production of cucurbita-5,24-diene-3β,23α-diol. A mild acid treatment of this compound resulted in an isomerization of the C23-OH group to C25-OH with the concomitant migration of a double bond, suggesting that a nonenzymatic transformation may account for the observed C25-OH in the majority of cucurbitacins found in plants. The functional expression of CYP88L7 resulted in the production of hydroxylated C19 as well as C5-C19 ether-bridged products. A plausible mechanism for the formation of the C5-C19 ether bridge involves C7 and C19 hydroxylations, indicating a multifunctional nature of this P450. On the other hand, functional CYP88L8 expression gave a single product, a triterpene diol, indicating a monofunctional P450 catalyzing the C7 hydroxylation. Our findings of the roles of several plant P450s in cucurbitacin biosynthesis reveal that an allylic hydroxylation is a key enzymatic transformation that triggers subsequent processes to produce structurally diverse products.

Keywords: Momordica charantia; allylic hydroxylation; biosynthesis; cucurbitacin; cytochrome P450; enzyme catalysis; multifunctional enzyme; plant; secondary metabolism; terpenoid.

Figure 1.

Biosynthetic pathway of cucurbitacins in M. charantia. Carbon numbering is shown in the structure of cucurbitadienol (1).

Figure 2.

A, a network module representing correlations of 18 contigs highly correlated with McCBS (blue circle, M01391) in the ConfeitoGUIplus analysis. Pink circles (M00873 (CYP88L8), M01465 (CYP81AQ19), and M04110 (CYP88L7)) indicate the candidate contigs annotated as P450 in the BLAST search. The annotation of 18 selected contigs is described in Table S2. Solid lines and dotted lines indicate correlations obtained by the FPO and the FNI analysis, respectively (26). B, RPKM (reads per kilobase per million mapped reads) values from the RNA-Seq analysis of three candidate P450 genes in each tissue, which were similar to those of McCBS. Red and black bars, McCBS and candidate P450 genes, respectively. OL, old leaves; YL, young leaves; St, stems; Te, tendrils; MF, male flowers; FF, female flowers; Fr, fruits; SL, seedling leaves; SS, seedling stems; SR, seedling roots.

Figure 3.

LC/MS-MS chromatogram of yeast extracts from the expression of empty vector, McCBS, McCBS/LjCPR/CYP81AQ19, McCBS/LjCPR/CYP88L7, and McCBS/LjCPR/CYP81AQ19/CYP88L7. For products 10 and 11, peaks could not be assigned due to an identical mass. Moreover, peaks for aldehyde 8 and 9 could not be found on the McCBS/LjCPR/CYP81AQ19/CYP88L7 chromatogram.

Figure 4.

CYP81AQ19 catalyzed hydroxylation at the C23 of cucurbitadienol. The allylic hydroxyl group underwent a dehydration to produce 3 and an isomerization to give 4 under acidic conditions.

Figure 5.

CYP88L7 catalyzed hydroxylation at C19 as well as the ether bridge formation between C5 and C19. Upon co-expression with CYP81AQ19, aldehyde products 8 and 9 were also identified.

Figure 6.

A, HPLC chromatogram of yeast extracts expressing empty vector (black) and McCBS/LjCPR/CYP88L8 (green). B, CYP88L8-catalyzed C7β hydroxylation of cucurbitadienol.

Figure 7.

Quantitative RT-PCR analysis of McCBS (blue; left), CYP81AQ19 (red; center), and CYP88L7 (green; right) mRNA levels in the leaves (L), stems (St), fruits (Fr), seedling leaves (SL), seedling stems (SS), and seedling roots (SR). McActin was utilized as a reference. Data represent mean ± S.D. (error bars) from three independent experiments.

Figure 8.

Allylic hydroxylation triggers further structural rearrangements. A, isomerization of C23-OH into C25-OH via allylic cation intermediate. B, ether bridge formation triggered by dehydroxylation of C7-OH.

Figure 9.

Amino acid sequence alignment between CYP88L7 and CYP88L8 from M. charantia. Putative substrate recognition sequences (SRS 1–6) are denoted with blue underlines (➀ to ➅) (36). The conserved heme binding sequence is indicated with a red underline.