PLANT STEROL BIOSYNTHESIS - IDENTIFICATION AND CHARACTERIZATION OF 2 DISTINCT MICROSOMAL OXIDATIVE ENZYMATIC SYSTEMS INVOLVED IN STEROL C4-DEMETHYLATION

Membrane-bound enzymatic systems obtained from maize embryos that cata lyze the oxidative C4-mono-demethylation of 4,4-dimethyl- and 4alpha-m ethylsterols have been investigated. Enzymatic assay conditions have b een developed for the first time to detect the C4-monodemethylated pro ducts formed. The properties of the microsomal systems have been estab lished for cofactor requirements and kinetics. The demethylation proce ss has been interrupted to demonstrate the formation of stable, oxygen ated intermediates. In addition to the 3-keto and 3beta-hydroxy-4-mono demethylated products formed, three new sterols have been identified. ergosta-9beta,19-cyclo-24(24(1))-en-4alpha-hydroxy methyl was identifi ed for the first time as the immediate metabolite of 24-methylenecyclo artanol by 4alpha-methyl oxidase in addition to droxy-4beta,14alpha-di methyl-5alpha-ergosta-9beta, 19-cyclo-24(24(1))-en-4alpha-carboxylic-a cid and stigmasta-7,24(24(1))-dien-4alpha-carboxylic-acid, intermediat es involved respectively in the oxidative demethylation of 24-methylen ecycloartanol and 24-ethylidenelophenol. Proton nuclear magnetic reson ance studies of enzymatically produced gosta-9beta,19-cyclo-24(24(1))e n-4alpha-carboxylic acid indicate that the 4alpha-methyl group of 24-m ethylenecycloartanol is oxidized and subsequently removed during its e nzymatic conversion to cycloeucalenol. From a series of incubations wi th 25 natural or synthetic 4,4-dimethyl and 4alpha-methylsterols, a hi gh degree of substrate specificity for the oxidation at C4 of 4,4-dime thyl- and 4alpha-methylsterols was determined. Our results indicate th at oxidation of the 4alpha-methyl group of the 4,4-geminal dimethylste rols requires the more flexible and presumably bent conformation of 9b eta,19-cyclopropylsterols and the absence of a DELTA24(25) unsaturatio n, whereas the rigid planar conformation of DELTA7-unsaturated sterols favors oxidation of 4alpha-methylsterols. Distinct strict structural requirements for the oxidation of 4,4-dimethyl- and 4alpha-methylstero ls and different sensitivity toward cyanide ions and 3beta,5alpha,6alp ha-stigmastatriol, a novel inhibitor of 4alpha-methylsterol C4 oxidase activity, are consistent with the conclusion that two distinct oxidat ive systems are involved in the removal of the first and second C4-met hyl group of phytosterol precursors. Moreover, the present study direc tly establishes that during the conversion of cycloartenol to phytoste rol one C4 dealkylation occurs before the removal of the 14alpha-methy l group.