phytosterols and obtusifoliol

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1β-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ

Topics: Australia; Cholesterol; Cytochrome P450 Family 51; Dioscorea; Oxidoreductases; Phytosterols; Saccharomyces cerevisiae; Saponins; Transcriptome

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, beta-sitosterol, Delta5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1,515 mg/100g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100g oil) was detected in H52 at 14 DAF. During linseed development, beta sitosterol (830 mg/100g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100g oil) and stigmasterol (265 mg/100g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.

Topics: Cholestadienols; Cholesterol; Chromatography, Thin Layer; Flax; Flowers; Gas Chromatography-Mass Spectrometry; Phytosterols; Seeds; Sitosterols; Species Specificity; Stigmasterol; Time Factors; Triterpenes

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

Topics: Arabidopsis; Arabidopsis Proteins; Cell Membrane; Cholestadienols; Cytochrome P-450 Enzyme System; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Molecular Sequence Data; Mutation; Oxidoreductases; Phenotype; Phytosterols; Seedlings; Sterol 14-Demethylase

Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein. The corresponding segment from the yeast lanosterol 14alpha-demethylase was linked to the partial wheat cDNA and the chimera expressed in Saccharomyces cerevisiae. Compared to microsomes from control yeasts, membranes of yeast expressing the chimera catalysed 14alpha-demethylation of obtusifoliol with an increased efficiency relative to lanosterol demethylase activity. W516 is thus a plant member of the most ancient and conserved P450 family, CYP51.

Topics: Amino Acid Sequence; Base Sequence; Cholestadienols; Cloning, Molecular; Cytochrome P-450 Enzyme System; DNA Primers; DNA, Complementary; Evolution, Molecular; Gene Library; Lanosterol; Microsomes; Molecular Sequence Data; Oxidoreductases; Phylogeny; Phytosterols; Polymerase Chain Reaction; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Sequence Homology, Amino Acid; Sterol 14-Demethylase; Substrate Specificity; Triticum

Analysis of the sterol compositions of 13 clinical isolates of the pathogenic yeast Cryptococcus neoformans obtained from five patients with recurring cryptococcal meningitis showed that, unlike Candida albicans, the major sterols synthesized by this yeast were obtusifoliol (range, 21.1 to 68.2%) and ergosterol (range, 0.0 to 46.5%). There was considerable variation in the sterol contents among the 13 isolates, with total sterol contents ranging from 0.31 to 5.9% of dry weight. The isolates from the five patients who had relapses had different total sterol contents and compositions in comparison with those of the pretreatment isolates, indicating either that the sterols had been changed by therapy or that the patients were infected with new isolates with different sterol compositions. Growth of the cryptococcal isolates in the presence of subinhibitory concentrations of fluconazole (0.25x the MIC) significantly altered the sterol content and pattern. The total sterol content decreased in nine isolates and increased in four isolates in response to pretreatment with fluconazole. Fluconazole had no consistent effect on ergosterol levels. In contrast, fluconazole caused a decrease in obtusifoliol levels and an increase in 4,14-dimethylzymosterol levels in all isolates. These results indicate extensive diversity in sterol content, sterol composition, and sterol synthesis in response to subinhibitory concentrations of fluconazole in C. neoformans strains. We propose that fluconazole inhibits the sterol synthesis of C. neoformans by interfering with both 14 alpha-demethylase-dependent and -independent pathways. No correlation between the sterol compositions of C. neoformans isolates and their susceptibilities to fluconazole was found.

Topics: Cholestadienols; Cryptococcus neoformans; Ergosterol; Fluconazole; Humans; Microbial Sensitivity Tests; Phytosterols; Sterols

A series of 7-oxo-obtusifoliol analogues have been synthetized and investigated as potential inhibitors of cytochrome P-450 dependent obtusifoliol 14 alpha-demethylase (P-450OBT.14DM) from higher plant microsomes. 7-Oxo-24 xi(24')-dihydro-obtusifoliol and 7-oxo-24(25)-dihydro-29-nor-lanosterol were potent competitive inhibitors for P-450OBT.14DM, binding 125-200 times more tightly than the substrates obtusifoliol and 24(25)-dihydro-29-nor-lanosterol. Inhibition of P-450OBT.14DM by these analogues showed strict structural requirements including the 8-en-7-one system which was compulsory for binding. 7-Oxo-24(25)-dihydro-lanosterol possessing an additional 4 beta-methyl substituent, did not have such inhibitory effects. Treatment of cultures of suspended bramble cells with 7-oxo-24(25)-dihydro-29-nor-lanosterol resulted in a strong decrease of [14C]acetate incorporation into the demethylsterols fraction and in an accumulation of [14C]obtusifoliol. This confirms that P-450OBT.14DM is the main in vivo target of 7-oxo-24(25)-dihydro-29-nor-lanosterol in the sterol-biosynthetic pathway.

Topics: Cells, Cultured; Cholestadienols; Cytochrome P-450 Enzyme Inhibitors; Eucalyptus; Hordeum; Lanosterol; Oxidoreductases; Phytosterols; Plants, Medicinal; Sterol 14-Demethylase; Suspensions; Zea mays

The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined. In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum. P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol. The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol. The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM. They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction. Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast. This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.

Topics: Cholestadienols; Cytochrome P-450 Enzyme System; Kinetics; Microsomes; Molecular Conformation; Molecular Structure; Oxidoreductases; Phytosterols; Sterol 14-Demethylase; Substrate Specificity; Zea mays

Two classes of polyene-resistant mutants were isolated from survivors of N-methyl-N'-nitro-N-nitrosoguanidine treatment of a wild-type Candida albicans. An analysis of the major sterols of one class revealed an accumulation of lichesterol and fecosterol while the other class accumulated eburicol, obtusifoliol, and lanosterol with minor quantities of C28 sterols.

Topics: Amphotericin B; Candicidin; Candida albicans; Cholestadienols; Drug Resistance, Microbial; Ergosterol; Lanosterol; Mutation; Natamycin; Nystatin; Phytosterols; Triterpenes