Cycloartenol-derived sterol biosynthesis in the Peronosporales

Abstract

Selected species of the order Peronosporales, which are unable to epoxidize squalene and thus synthesize sterols, are able to metabolize exogenous cycloartenol to lanosterol and in some organisms to fucosterol, ergosterol, and cholesterol. Lanosterol was less effectively utilized but some ergosterol and cholesterol were yielded. Fucosterol was very efficiently metabolized by most species to ergosterol, A7-ergostenol, A5-ergostenol, cholestanol, and cholesterol. Several unknown sterols were observed in most trials. These data suggest a vestigial sterol synthetic pathway derived from cycloartenol, followed by possible isomerization to lanosterol and then to other sterols.It has been established that species of Pythium and Phytophthora require exogenous sterols for sexual reproduction (1, 2). These organisms are unable to synthesize sterols because of a missing squalene epoxidase (3). Metabolism of exogenous sterols has been observed in Phytophthora cactorum by Langeake (4), who reported possible conversion of lanosterol to cholesterol. Nes and Patterson (5) reported that Phyt. cactorum was unable to transform cycloartenol or lanosterol to other sterols. Conversion ofA7 and A5'7 sterols to A5 sterols has been reported by Elliott and Knights (6) in Phyt. cactorum, which additionally synthesizes cholesteryl esters, cholesteryl glucosides, and acyl sterol glycosides (7,8). Other than the above, sterol conversions in Phytophthora or Pythium have, in general, not been demonstrated in detail.Lagenidium giganteum is another Oomycete, but of the Lagenidiales, that requires exogenous sterols to produce zoospores (9). Warner and Domnas (10) have shown that this organism converts cycloartenol, but not lanosterol, to cholesterol. Because Lagenidium species are able to metabolize cycloartenol, it was ofconsiderable interest to observe ifthis compound could be metabolized by selected species ofthe Peronosporales. This article reports our observations. Inoculation and growth were as described (10) (10) and the unsaponifiable lipid fraction was obtained. The sterols were separated on HPLC with a Waters instrument by using a 10-,um C18 column (4.6 x 250 mm). Fractions were eluted with methanol/H20 (96:4) at 2 ml/min flow with UV detection at 210 nm. MATERIALS AND METHODS MicroorganismsSterols were identified by gas/liquid chromatography on 3% SE-30 and 1% QF-1 with a Packard 417 FID chromatograph as described (10). Confirmation ofidentification was obtained with gas chromatography/electron impact mass spectroscopy (70 eV) with a Finnegan 4023 instrument. A 15-m flexible fused silica capillary coated with OV-101 was programmed from 160 to 2600C at 10°C/min with an initial hold of 3 min. RESULTSNo sterols were found in control organisms to the limit of detection (0.0001%). Uninoculated control cultures yielded a stoichiometric recovery of the administered sterols.Sterols Isolated After Administration ofCycloartenol. Table 1 illustrates the substances identified in the organisms examined. Clear evidence was obtained for the ...

Cell Wall Polysaccharide Synthases Are Located in Detergent-Resistant Membrane Microdomains in Oomycetes

“…Thus, unlike parasitic Peronosporales such as Pythium and Phytophthora species, which are unable to epoxidize squalene and consequently synthesize sterols (72), S. monoica is able to produce its own pool of sterols without requiring any exogenous sterol. This observation and the occurrence of fucosterol in S. monoica are consistent with the cyclization of 2,3-oxidosqualene to cycloartenol and thus with the biosynthesis of sterols through the cycloartenol pathway in Saprolegniales (72).…”

“…Consistent with the early suggestion that oomycetes should be grouped with brown algae rather than with true fungi on the basis of their sterol composition (13), the plasma membranes and DRMs from S. monoica were devoid of ergosterol and the corresponding unsaturated derivatives, which are the major sterols of true fungi and products of the lanosterol pathway (46,72). None of the ⌬ 5 -sterols that are major sterols in DRMs from higher plants, such as sitosterol and stigmasterol (6,39,49), were present in detectable amounts in the purified DRMs from S. monoica.…”

Evidence for metabolic and functional discrimination of sterols by Phytophthora cactorum

“…Because [14C]mevalonate was not converted to labeled sterol the investigators suggested the sterol was "almost certainly due to cholesterol from the original slope of P. cactorum being transferred to the liquid cultures." The sterols observed in the former studies (5,(34)(35)(36) may arise in a similar fashion from the residual agar, which was coextracted with the harvested mycelium. When the agar was extracted with acetone and then used to solidify the medium, as expected, no oogonia or oospores were observed in 21-day control cultures and no detectable sterols were present in the mycelium.…”

“…In the metabolism studies, to verify structure identities and quantities, two additional packed columns were used-i.e., 1% XE-60 and 1% SP-1000 (Alitech Associates, Deerfield, IL, and Supelco, Bellefonte, PA, respectively)-which were operated at 200 and 2550C, respectively. In some cases either 100 ,ug of stigmasterol [retention time relative to cholesterol (RRT) 1 (5,(34)(35)(36) or presumably are formed by metabolism of some appropriate 4,4,14-trimethylsterol precursor (5,36). However, the identification in these cases was not supported by the use of radiolabeled acetate, mevalonate, squalene, or lanosterol (8-10, 27, 28).…”

“…The occurrence, biosynthesis, and metabolism of sterols has been studied in numerous oomycetous fungi [orders Saprolegniales (1-3), Leptomitales (1), Lagenidiales (4), and Peronosporales (5)(6)(7)(8)(9)(10)]. The physiological roles of sterols and other polycyclic isopentenoids in effecting growth and reproduction have also been the subject of much investigation (11)(12)(13)(14).…”