4-methyl zymosterone and other 4-methyl intermediates of sterol biosynthesis from yeast mutants engineered in the ERG27 gene encoding 3-ketosteroid reductase

Studies in the post-squalene section of sterol biosynthesis may be hampered by the non-availability of authentic standards[1]. The present study used a series of yeast strains engineered in 3-ketosteroid reductase (Erg27p) to obtain radioactive and non-radioactive intermediates non-commercially available of sterol biosynthesis. Some of these strains had been used in previous studies[2]. Non-radioactive 3-keto 4-monomethyl sterones were purified from nonsaponifiable lipids extracted from cells bearing point-mutated 3-ketosteroid reductase. TLC silicagel chromatography gave a fraction (named Keto 1) which resulted to be (GLC-MS analysis) a mixture of 4-methyl zymosterone and 4-methyl fecosterone. The two sterones were successfully separated by AgNO3 TLC chromatography. Two strategies were adopted to prepare the radioactive compounds: i) incubation of cell homogenates of an ERG27-deletant strain with radioactive lanosterol; ii) incubation of growing cells of a strain expressing point-mutated 3-ketosteroid reductase with radioactive acetate. Chemical reduction with NaBH4 of both radioactive and non-radioactive 3-keto sterones produced the physiological 3-beta OH sterols, as well as the non-physiological 3-alpha OH isomers. The above 4-methyl steroids, both radioactive and non-radioactive, will enable us to carry out a series of experiments aimed at characterizing enzymes of the C4 –demethylase complex of sterol biosynthesis. Furthermore, the reported toxicity of such compounds toward the Hedgehog proteins[3] can now be assayed directly. Preliminary experiments with 3-OH -alpha and 3–beta methyl zymosterol suggested that the non-physiological alpha isomer could be used as inhibitor of C4-demethylase complex. References [1] J. Ačimovič, D. Rozman, Molecules 2013, 18, 4002-4017. [2] B. Teske, S. Taramino, M. S. A. Bhuiyan, N. S. Kumaraswami, S. K. Randall, R. Barbuch, J. Eckstein, G. Balliano, M. Bard, Biochim. Biophys. Acta 2008, 1781, 359-366. [3] R. W. Stottmann, A. Turbe-Doan, P. Tran, L. E. Kratz, J. L. Moran, R. I. Kelley, D. R. Beier, PLoS Genet. 2011, 7, 1-16.