Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae

Indiana University-Purdue University Indianapolis, Biology Department, Indianapolis, IN 46202, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 06/2008; 1781(8):359-66. DOI: 10.1016/j.bbalip.2008.04.017
Source: PubMed


Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.

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Available from: Stephen K. Randall, Oct 01, 2015
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    ABSTRACT: In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.
    Biochimica et Biophysica Acta 10/2009; 1801(2):156-62. DOI:10.1016/j.bbalip.2009.10.005 · 4.66 Impact Factor
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    ABSTRACT: In mammals and yeasts, oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol, the first cyclic intermediate in sterol biosynthesis. We used a murine myeloma cell line (NS0), deficient in the 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7), as a model to study the potential interaction of the HSD17B7 with the OSC in mammals. HSD17B7 is the orthologue of the yeast steroid-3-ketoreductase (ERG27), an enzyme of ergosterol biosynthesis that plays a protective role towards OSC. Tracer experiments with NS0 cells showed that OSC is fully active in these mammalian cells, suggesting that in mammals the ketosteroid reductase is not required for OSC activity. Mouse and human HSD17B7 were overexpressed in ERG27-deletant yeast cells, and recombinant strains were tested for (i) the ability to grow on different media, (ii) steroid-3-ketoreductase activity, and (iii) OSC activity. Recombinant strains grew more slowly than the control yeast ERG27-overexpressing strain on sterol-deficient media, whereas the growth rate was normal on media supplemented with a 3-ketoreductase substrate. The full enzymatic functionality of mammalian steroid-3-ketoreductase expressed in yeast along with the lack of (yeast) OSC activity point to an inability of the mammalian reductase to assist yeast OSC. Results demonstrate that in mammals, unlike in yeast, OSC and steroid-3-ketoreductase are non-interacting proteins.
    Biochimica et Biophysica Acta 11/2010; 1801(11):1232-7. DOI:10.1016/j.bbalip.2010.07.006 · 4.66 Impact Factor
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    ABSTRACT: In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain can function in the presence of the mammalian 3-keto reductase enzyme and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.
    Biochimica et Biophysica Acta 09/2012; 1831(2). DOI:10.1016/j.bbalip.2012.09.012 · 4.66 Impact Factor
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