Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative...

Conditions have been identified which permit metabolic formation of the third oxidized intermediate in the lanosterol 14 alpha-methyl demethylase reaction cascade. Metabolism of either the immediate precursor substrate 3 beta-hydroxylanost-8-en-32-al or lanost-8-ene-3 beta,32-diol under mixed function oxidase conditions affords formation of the intermediate. It must be emphasized that the intermediate can only be detected if saponification procedures are omitted during sterol isolation. Comparative chemical and biochemical studies of the isolated metabolite with 3 beta,15 alpha-dihydroxylanost-8-en-32-al reveal that the metabolite is not the 15 alpha-hydroxylanosterol aldehyde, a putative demethylase intermediate. The metabolite is efficiently converted to the demethylated delta 8,14-diene sterol in the absence of molecular oxygen or NADPH, thus supporting its identity as the final oxidized intermediate in the lanosterol 14 alpha-methyl demethylase cascade. 1H NMR analysis shows a proton resonance at 7.86 ppm consistent with a formyloxy proton. Mass spectral and infrared analysis of the metabolite clearly establish oxygen insertion into the immediate precursor substrate, 3 beta-hydroxylanost-8-en-32-al. Collectively, the biochemical and chemical characteristics of the metabolite support a structural assignment for the metabolite as 14 alpha-formyloxy-lanost-8-en-3 beta-ol.