The present study highlights differences between yeast Saccharomyces cerevisiae
and mammals regarding the interaction of the steroid-3-ketoreductase with oxidosqualene cyclase, two essential enzymes of sterol biosynthesis. In the yeast Saccharomyces cerevisiae
] (as well as in Candida albicans
] and Candida glabrata
[J. Whybrew and M. Bard, unpublished]), the absence of the 3-ketoreductase completely abolishes the functionality of oxidosqualene cyclase. By studying sterol biosynthesis in NS0 cells, a mouse cell line auxotrophic for cholesterol, specifically deficient in 3-ketoreductase activity [3
], we observed that in these mammalian cells oxidosqualene cyclase is active. Both tracer experiments and lipid analysis led to this conclusion. In cells incubated with radiolabeled acetate, no oxido- or dioxidosqualene (indicative of low or absent oxidosqualene cyclase activity) accumulated, whereas a 3-keto-4-monomethylsteroid accumulated at significant levels (50 to over 60% in the nonsaponifiable extracts, ). A ketosteroid compound was also the main component that accumulated in the GC-MS analyzed nonsaponifiable fraction extracted from NS0 cells (). Overall, these results suggest that the OSC is active in NS0 cells, thus the protective role of the 3-ketoreductase with respect to oxidosqualene cyclase activity is not conserved in mammals. This conclusion poses an intriguing question regarding the divergent evolution of the steroid-3-ketoreductase enzymes in yeast and mammals. Marijanovic et al. [7
] reported that both human and mouse 3-ketoreductase genes complemented the yeast ERG27
deletion and found that transformants expressing the mammalian enzymes grew much more slowly than a strain expressing the yeast ERG27 gene. We repeated these complementation experiments with new yeast recombinant strains overexpressing mammalian 3-ketoreductases (lacking, in our case, GST reporter group) and confirmed that the yeast ERG27 gene complements much better than mouse or human gene. Marijanovic et al. suggested that the slower growth was due to low expression of the mammalian enzyme in a yeast system. However, our data prove that the mammalian enzymes are catalytically active in yeast and complement the enzymatic function of Erg27p, allowing normal growth in media supplemented with lanosterol or 3-ketosteroids. Conversely, OSC is inactive in the yeast strains transformed with the human or mouse 3-ketoreductases, resulting in a severe growth defect. Therefore, in these recombinant strains, the poor protection of oxidosqualene cyclase rather than the low expression of mammalian proteins could explain the slow growth.
The failure of mammalian 3-ketoreductase to fulfill the role of protecting Erg7p in yeast suggests that in passing from yeasts to mammals these enzymes maintained the catalytic function, but lost the chaperone-like properties. What may account for the acquired independence of mammalian oxidosqualene cyclase from 3-ketoreductase? Comparison of the catalytic properties of yeast Erg27p and mammalian HSD17B7 enzymes allows speculation about the evolution of the 3-ketoreductase. The mammalian enzyme has previously been described as catalyzing also the conversion of steroid estrone to estradiol, thus playing a key role both in cholesterol synthesis and in steroid hormone synthesis. Breitling et al. [6
] suggested that mammalian enzyme (HSD17B7) is derived from proteins that probably reduced zymosterone or a similar compound to the corresponding alcohols and has only recently acquired its additional function in estradiol synthesis. This acquisition might have been accomplished by the loss of other functions such as the protective function towards the OSC. To support this speculation, additional experiments with novel yeast recombinant strains are necessary. For example, we do not know if the mammalian oxidosqualene cyclase maintains its independence when expressed in an erg27
yeast strain. If not, it would mean that the yeast background strongly influences the interaction between oxidosqualene cyclase and 3-ketoreductase.
In this case, it would be interesting to establish which is the best protector of mammalian cyclase expressed in yeast: whether the yeast 3-ketoreductase, which is closer to the cellular background, or the mammalian 3-ketoreductase, which is phylogenetically closer to mammalian oxidosqualene cyclase.
Likely, a more conclusive explanation of the evolutionary relationship between the OSC cyclase and 3-ketoreductase will come from extending the gene disruption approach to a wider series of organisms. Among these, two species may be particularly interesting: the yeast Schizosaccharomyces pombe
and the plant pathogenic fungus Botrytys cinerea
. S. pombe
shares some of the features of mammalian cells with regard to sterol synthesis such as the finding that the sterol C-4 demethylase complex is involved in the adaptation to low oxygen [26
]. B. cinerea
is responsible for grey mold diseases and its 3-ketoreductase has been recently recognized as the specific target of fenhexamid, one of the latest antibotrytis fungicides [28
]: would the inhibition of 3-ketoreductase affect OSC activity? Finally, the absence of interaction between mammalian 3-ketoreductase (HSD17B7) and oxidosqualene cyclase suggests that it is worth considering the protein interaction in fungal cells as a potential target for the development of specific antifungal drugs able to disrupt the yeast interaction even if they didn't inhibit 3-ketoreductase itself.
In NS0 cells, unlike in yeast, 3-ketoreductase does not assist oxidosqualene cyclase
Mouse and human 3-ketoreductases are catalytically active in yeast
Mouse and human 3-ketoreductases don't assist yeast oxidosqualene cyclase
When, from yeasts to mammals, the reductase- cyclase interaction, was lost?