Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation

The last unidentified gene encoding an enzyme involved in ergosterol biosyn thesis in Saccharomyces cerevisiae has been cloned. This gene, designated E RG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 s terol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catal yzes the sequential removal of the two methyl groups at the sterol C-4 posi tion. We developed a strategy to isolate a mutant deficient in converting 3 -keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize st erol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergosta diene and cholesterol, A new ergosterol auxotroph unable to grow on 3-ketos terols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disru ption failed to grow on various types of 3-keto sterol substrates. Surprisi ngly, when erg27 was grown on cholesterol- or ergosterol-supplemented media , the endogenous compounds that accumulated were noncyclic: sterol intermed iates (squalene, squalene epoxide, and squalene dioxide), and there was lit tle or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol tan upstream inter mediate of the C-4 demethylation process) and cholesterol tan end-product s terol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone , 4-methylfecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fif th intermediate was isolated and identified by H-1 NMR as a 4-wmethyl-24,25 -epoxy-cholesta-7-en-3-one. implications of these results are discussed.