Plant sterol biosynthesis. Identification and characterization of two distinct microsomal oxidative enzymatic systems involved in sterol C4-demethylation.
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.
Département d'Enzymologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique-Unité Propre de Recherche 406, Institut de Botanique, Strasbourg, France.
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D007202:Indicators and Reagents; D007700:Kinetics; D013058:Mass Spectrometry; D008861:Microsomes / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D015394:Molecular Structure; D013261:Sterols / Q000096:biosynthesis / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity; D014316:Tritium; D003313:Zea mays / Q000201:enzymology*
D007202:Indicators and Reagents; D013261:Sterols; D014316:Tritium; D006899:Mixed Function Oxygenases; C027532:methylsterol monooxygenase