Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

Abstract

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1⌬ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterolsupplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4␤,14␣-dimethyl-4␣-carboxy-cholesta-8,24-dien-3␤-ol and 4␤-methyl-4␣-carboxy-cholesta-8,24-dien-3␤-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

Steroids, triterpenoids and molecular oxygen

“…(ii) C-4 demethylases Demethylation of a C-4 methyl group requires the action of a suite of three enzymes working sequentially (Gachotte et al 1998(Gachotte et al , 1999Benveniste 2004):…”

The Effect of the erg26-1 Mutation on the Regulation of Lipid Metabolism in Saccharomyces cerevisiae

“…1A). ERG26 is believed to encode for the 4␣-CD activity in S. cerevisiae (17). Linkage analysis was performed in this mutant, designated H21, and showed that the mutation responsible for the ts defect resided within the ERG26 gene.…”

“…These strains have been very useful in determining the changes in sterol composition due to the individual loss of function of these genes. Yeast strains lacking the ERG25 gene accumulate the substrate of the Erg25p, 4,4-dimethylzymosterol (16), whereas strains lacking ERG26 accumulate the expected 4-methyl carboxy substrate intermediates (17). On the other hand, mutants lacking ERG27 function accumulate very little cyclic sterols (18).…”

“…In S. cerevisiae, the C-4 methyl oxidase, 4␣-CD, and C-3 ketoreductase required for the demethylation of 4,4-dimethylzymosterol are encoded for by the ERG25 (16), ERG26 (17), and ERG27 (18) genes, respectively. All three genes are essential under aerobic growth conditions.…”

Obtenção de levedura híbrida fluorescente e resistente a nistatina

“…A via biossintética do ergosterol inicia no esqualeno e depende de uma seqüência de 15 reações enzimáticas, cujos genes foram clonados e identificados (GACHOTTE et al,1998;GACHOTTE et al,1999). Esta via biossintética compreende a transformação do esqualeno em lanosterol, que é posteriormente convertido em zimosterol, o qual é transportado para dentro da mitocôndria, onde é metilado e origina o fecosterol.…”