Alternative pathways of sterol synthesis in yeast: Use of C 27 sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Abstract

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the Δ 8–Δ 7 isomerase (Erg2p) or Δ 5 desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5α-cholest-8-en-3β-ol, 8-dehydrocholesterol (Δ 5,8 sterol), or isodehydrocholesterol (Δ 6,8 sterol), together with the corresponding 3 α- 3 H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven Δ 22E sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag +-HPLC) and identified by gas chromatography–mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag +-HPLC. When Δ 8–Δ 7 isomerization was blocked, exogenous Δ 8 sterol underwent desaturation to Δ 5,8, Δ 6,8, and Δ 8,14 sterols. Formation of Δ 5,8 sterol was strongly favored over Δ 6,8 sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The Δ 5,8 sterol was metabolically almost inert except for Δ 22 desaturation, whereas the Δ 6,8 sterol was readily converted to Δ 5,7, Δ 5,7,9(11), and Δ 7,9(11) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, Δ 5,7 sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of Δ 5,8 and Δ 5,7,9(11) sterols in wild-type yeast compared to Smith–Lemli–Opitz individuals.