Publications

• Functional identification of sterol-4alpha-methyl oxidase cDNAs from Arabidopsis thaliana by complementation of a yeast erg25 mutant lacking sterol-4alpha-methyl oxidation.

  Darnet S., Bard M., Rahier A.

  Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identit ... >> More

  Specific primers derived from both genomic sequence data and EST cDNA sequences were used to polymerase chain reaction amplify two full-length cDNA sequences (AtSMO1 and AtSMO2), 801 and 783 bp, respectively, from an Arabidopsis thaliana cDNA library. The predicted proteins show 32 and 29% identity to the ERG25 gene from Saccharomyces cerevisiae which encodes the sterol-4alpha-methyl oxidase (SMO), a membrane-bound non-heme di-iron oxygenase involved in lipid metabolism. Heterologous expression of AtSMO1 and AtSMO2 in a yeast erg25 ergosterol auxotroph, lacking SMO activity, restored growth and endogenous ergosterol synthesis. These results represent the first functional identification of SMO genes from plants. << Less

  FEBS Lett. 508:39-43(2001) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Plant sterol biosynthesis. Identification and characterization of two distinct microsomal oxidative enzymatic systems involved in sterol C4-demethylation.

  Pascal S., Taton M., Rahier A.

  Membrane-bound enzymatic systems obtained from maize embryos that catalyze the oxidative C4-monodemethylation of 4,4-dimethyl- and 4 alpha-methylsterols have been investigated. Enzymatic assay conditions have been developed for the first time to detect the C4-monodemethylated products formed. The ... >> More

  Membrane-bound enzymatic systems obtained from maize embryos that catalyze the oxidative C4-monodemethylation of 4,4-dimethyl- and 4 alpha-methylsterols have been investigated. Enzymatic assay conditions have been developed for the first time to detect the C4-monodemethylated products formed. The properties of the microsomal systems have been established for co-factor requirements and kinetics. The demethylation process has been interrupted to demonstrate the formation of stable, oxygenated intermediates. In addition to the 3-keto and 3 beta-hydroxy-4-monodemethylated products formed, three new sterols have been identified. 3 beta-Hydroxy-4 beta,14 alpha-dimethyl-5 alpha-ergosta-9 beta,19-cyclo-24(24(1))-en-4 alpha-hydroxy methyl was identified for the first time as the immediate metabolite of 24-methylenecycloartanol by 4 alpha-methyl oxidase in addition to 3 beta-hydroxy-4 beta,14 alpha-dimethyl-5 alpha-ergosta-9 beta,19-cyclo-24(24(1))-en-4 alpha-carboxylic-acid and 3 beta-hydroxy-5 alpha-stigmasta-7,24(24(1))-dien-4 alpha-carboxylic-acid, intermediates involved respectively in the oxidative demethylation of 24-methylenecycloartanol and 24-ethylidenelophenol. Proton nuclear magnetic resonance studies of enzymatically produced 3 beta-hydroxy-4 beta,14 alpha-dimethyl-5 alpha-ergosta-9 beta,19-cyclo-24(24(1))en-4 alpha-carboxylic acid indicate that the 4 alpha-methyl group of 24-methylenecycloartanol is oxidized and subsequently removed during its enzymatic conversion to cycloeucalenol. From a series of incubations with 25 natural or synthetic 4,4-dimethyl and 4 alpha-methylsterols, a high degree of substrate specificity for the oxidation at C4 of 4,4-dimethyl- and 4 alpha-methylsterols was determined. Our results indicate that oxidation of the 4 alpha-methyl group of the 4,4-geminal dimethylsterols requires the more flexible and presumably bent conformation of 9 beta,19-cyclopropylsterols and the absence of a delta 24(25) unsaturation, whereas the rigid planar conformation of delta 7-unsaturated sterols favors oxidation of 4 alpha-methylsterols. Distinct strict structural requirements for the oxidation of 4,4-dimethyl- and 4 alpha-methylsterols and different sensitivity toward cyanide ions and 3 beta,5 alpha,6 alpha-stigmastatriol, a novel inhibitor of 4 alpha-methylsterol C4 oxidase activity, are consistent with the conclusion that two distinct oxidative systems are involved in the removal of the first and second C4-methyl group of phytosterol precursors. Moreover, the present study directly establishes that during the conversion of cycloartenol to phytosterol one C4 dealkylation occurs before the removal of the 14 alpha-methyl group. << Less

  J Biol Chem 268:11639-11654(1993) [PubMed] [EuropePMC]

  This publication is cited by 13 other entries.

• Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases.

  Darnet S., Rahier A.

  In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct fam ... >> More

  In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4alpha-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9beta,19-cyclopropylsterols (i.e. 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4alpha-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4alpha-methyl-Delta7-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4alpha-methylsterol precursors respectively. << Less

  Biochem. J. 378:889-898(2004) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• The role of cytochrome b5 in 4alpha-methyl-oxidation and C5(6) desaturation of plant sterol precursors.

  Rahier A., Smith M., Taton M.

  The electron donors for the membrane-bound sterol-4alpha-methyl-oxidases and sterol C5(6)-desaturase of plant sterol biosynthesis have not been previously identified. The requirement of cytochrome b5 to shuttle reducing equivalents from NAD(P)H to 4,4-dimethylsterol-4alpha-methyl oxidase (4,4-DMSO ... >> More

  The electron donors for the membrane-bound sterol-4alpha-methyl-oxidases and sterol C5(6)-desaturase of plant sterol biosynthesis have not been previously identified. The requirement of cytochrome b5 to shuttle reducing equivalents from NAD(P)H to 4,4-dimethylsterol-4alpha-methyl oxidase (4,4-DMSO), 4alpha-methylsterol-4alpha-methyl oxidase (4alpha-MSO), and delta7-sterol-C5(6) desaturase (5-DES) was investigated using a purified preparation of IgG raised against plant cytochrome b5. The activities of 4,4-DMSO, 4alpha-MSO, and 5-DES, three oxidative reactions not mediated by cytochrome P-450, were strongly and completely inhibited by the antibody in a microsomal preparation from maize. In addition the IgG also inhibited NADH-dependent cytochrome c reduction in the same preparation. These results strongly suggest that membrane-bound cytochrome b5 of maize microsomes is an obligatory electron carrier from NAD(P)H to 4,4-DMSO, 4alpha-MSO, and 5-DES. << Less

  Biochem Biophys Res Commun 236:434-437(1997) [PubMed] [EuropePMC]

  This publication is cited by 10 other entries.

• Dissecting the sterol C-4 demethylation process in higher plants. From structures and genes to catalytic mechanism.

  Rahier A.

  Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it ... >> More

  Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it does in yeast and animals, but is interrupted by several enzymatic steps. Each C-4 demethylation step involves the sequential participation of three individual enzymatic reactions including a sterol methyl oxidase (SMO), a 3β-hydroxysteroid-dehydrogenase/C4-decarboxylase (3βHSD/D) and a 3-ketosteroid reductase (SR). The distant location of the two C-4 demethylations in the sterol pathway requires distinct SMOs with respective substrate specificity. Combination of genetic and molecular enzymological approaches allowed a thorough identification and functional characterization of two distinct families of SMOs genes and two 3βHSD/D genes. For the latter, these studies provided the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants, and the first data on 3-D molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthetic pathway with its substrate in animals, yeast and higher plants. Characterization of these three new components involved in C-4 demethylation participates to the completion of the molecular inventory of sterol synthesis in higher plants. << Less

  Steroids 76:340-352(2011) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.