Campesterol biosynthesis

The Arabidopsis mutant dwf7/ste 1 is defective in C5-desaturation of episterol (Fig. (2)) [18], thus impaired in an enzyme function involved in a very early step of brassinosteroid precursor biosynthesis. The enzymatic block of dwf7/stel was determined by feeding experiments using l3C-labelled mevalonic acid and a subsequent analysis of endogenous sterol and brassinosteroid precursors. The mutant accumulates episterol with a simultaneous decrease of downstream intermediates (24-methylenecholesterol, campesterol, castasterone, brassinolide). 

The demonstration166 that the side-chains of 24-methylenecycloartanol (96), 24-methylenelophenol, and campesterol incorporated two deuterium atoms, whilst those of 24-ethylidenecycloartanol, stigmasterol, and sitosterol contained a maximum of four deuterium atoms when biosynthesized by cultures of barley embryo in the presence of [Me-2H3]methionine, provides further evidence for the intermediacy of 24-methylene- and 24-ethylidene-compounds in the biosynthesis of C28 and C29 phytosterols. The barley system was also able to convert labelled 24-ethylidenelophenol into radioactive sitosterol efficiently. These results, and those obtained from feeding experiments with [2-14C,(4R)-4-3Hi]MVA, are consistent with a pathway (Scheme 9) to stigmasterol (97) involving isomerization of (98) to a... 

Figure 6.10 Biosynthesis of Brassinolide from campesterol. Enzymes identified from Arabidopsis thaliana are shown. Enzymes from other plants are identified in the text (Fujioka and Yokota, 2003).

Sterols include 4a-methylsterols, intermediates of sterols and triterpens biosynthesis. Analysis of the sterol fraction demostrated the constant presence of P-sitosterol, campesterol and stigmasterol, A -avenasterol, while A" -avenasterol may be absent or present in very low amount. P-sitosterol makes up the 75-90% of the total sterol fraction, the rest of sterols occur in minute quantities. 

The structural relationship between phytosterols and BRs has been proposed from the biosynthetic points of view. All naturally occurring BRs possess carbon skeletons identical to those of common phytosterols (e.g., campesterol, 24-methylene-cholesterol, isofiicosterol, sitosterol, and cholesterol). Thus, BRs may be speculatively regarded as the enzymatic oxidation products of phytosterols with the corresponding carbon skeletons, as is the case of the biosynthesis of other steroid hormones (e.g., ecdysteroids (25) and 1,25-dihydroxyvitamin D3 (26)). Although BL has recently been proved to be biosynthesized from CS in crown gall cells of Catharanthus roseus (27), a major part of the biosynthesis of BRs is remained to be investigated. Experiments using radio-labeled precursors are required to clarify the biosynthesis of BRs in a suitable plant system. 

Fig. 2. Biosynthesis of typical plant sterols. Boxed sterols are probable precursors of various brassinosteroids. Among these sterols, sitosterol, stigmasterol, campesterol, 24-epicampesterol and cholesterol are the most common end-of-pathway sterols in plants. Italic letters refer to the lesions in the biosynthesis mutants of Arabidopsis (dim) and pea (Ikb).

Fig. 3. Biosynthesis of brassinolide from campesterol. Brassinolide is synthesized from castasterone which is derived from campestanol by either late C6-oxidation pathway or early C6-oxidation pathway. Campesterol is converted to campestanol by hydrogenation. Italic letters refer to the lesions in the biosynthesis and sensitivity mutants of Arabidopsis det2, dwf4, cpd and bril) and pea (Ik and Ika). The asterisked pathways and compounds are hypothetical.

Fig. 4. Multi-step biosynthesis of campesterol to campestanol. The biosynthetic lesion of the det2 mutant of Arabidopsis is shown.

BRs belong to the class of molecules known as triterpenoids. Because BRs are a group of modified sterols, the BR biosynthetic pathway can be divided into two major parts the sterol-specific pathway (which converts squalene to campesterol) and the BR-specific pathway (which converts campesterol to the BR). In the sterol-specific pathway, mevalonic acid (which is the starting material in terpenoid biosynthesis) is condensed and cyclized to produce 2,3-oxidosqualene. This precursor is further modified to form the major plant sterols such as sitosterol and campesterol. To become bioactive BRs, sterols must be processed by the BR-specific pathway. 

In D. tertiolecta, we were able to idenhfy the majority of enzymes responsible for the biosynthesis of stigmasterol and p-sitosterol. We have also identified the enzymes synthesizing episterol, a precursor of campesterol, which is an important phytosterol known to have potential antiosteoarthritic properties (Gabay et al., 2010). Episterol is also a precursor for ergosterol however, the biosynthetic pathway branches after (S)-squalene-2,3-epoxide formation and follows a different route. Due to the fact that we did not identify most of the... 

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