Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in tissues of the ruminants, Sus scrofa and Homo sapiens is involved in biosynthesis of steroid hormones, catalyzing a double-bond isomerization reaction as the last step of synthesis of Δ⁴-pregnene-3,20-dione (progesterone) and the obligatory step in the synthesis of the last precursor of testosterone, Δ⁴-androstenene-3,17-dione. As neurosteroids, steroid hormones are involved in such diverse functions as cognition, depression and memory and are suggested to play a protective role in neuropathologies including Alzheimer’s disease, Parkinson’s disease and brain injury.

The human GST A3-3 is the most efficient steroid double-bond isomerase known so far in mammals. The current work extends discoveries of GST enzymes that act in the steroidogenic pathways in large mammals to Equus ferus caballus. In contrast to the rodents, Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes it a more suitable model for human steroidogenesis than the murine one.

In the present study, the mRNA encoding the steroid isomerase GST A3-3 was cloned from stallion testis. The equine GST A3-3 (EcaGST A3-3) was heterologously expressed in E. coli and purified by centrifugation, sonication, affinity chromatography and dialysis. The in vitro measurements of enzymatic activity were followed spectrophotometrically and revealed highly efficient steroid double-bond isomerase activity in the biosynthetic pathways to progesterone and testosterone. The enzyme now ranks as one of the most efficient steroid isomerases known in mammals.

The concentrations of EcaGSTA3 mRNA were highest in hormone-producing organs such as ovary, testis and adrenal gland. The high efficiency and the tissue distribution of EcaGST A3-3 support the view that the enzyme plays a physiologically significant role in the biosynthesis of steroid hormones.

Inhibition of EcaGST A3-3 might help treat reproductive and neurodegenerative disorders. An FDA-approved library of 1040 compounds was screened for novel inhibitors of EcaGST A3-3. The inhibition pattern of EcaGST A3-3 is similar to that of the human GST A3-3.

Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication. However, some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in tissues of Homo sapiens (humans), Sus scrofa (pigs) and ruminants is involved in biosynthesis of steroid hormones, catalyzing a double-bond isomerization reaction as the last step of synthesis of Δ⁴-pregnene-3,20-dione (progesterone) and the obligatory step in the synthesis of the last precursor of testosterone, Δ⁴-androstenene-3,17-dione. Steroids regulate several vital aspects of life such as for example glucose homeostasis, inflammation, immunosuppression, blood pressure, reproduction and pregnancy.

The human GST A3-3 was the most efficient steroid double-bond isomerase known so far in mammals. Our work extends discoveries of GSTs that act in the steroidogenic pathways in large mammals to Equus ferus caballus (horse). The kinetic profile of EcaGST A3-3 reveals a catalytic efficiency higher than that of the human enzyme making EcaGST A3-3 the most efficient steroid double-bond isomerase known today in mammals.

In contrast to the rodents, Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes it a more suitable model for human steroidogenesis than the murine one. Inhibition of EcaGST A3-3 might help treat endocrine disorders. We screened a library of 1040 FDA-approved compounds for novel inhibitors of EcaGST A3-3 and made a further characterization of the most potent inhibitors.

To extend the search for steroidogenic GSTs to other mammals, we probed the degree of GST A3-3 amino acid sequence conservation in Homo sapiens, Equus ferus caballus, Canis lupus familiaris (dog), Capra hircus (goat) and Monodelphis domestica (gray short-tailed opossum). We generated expression vectors containing homologous DNA from these species to facilitate further evaluation of the activity of these GSTs in mammals.

We continued to expand the research to insects by investigating the steroidogenic activity of GSTE14 in Drosophila melanogaster (fruit fly), where this enzyme has been shown to be implicated in molting.

Our work has provided insights into the role of GSTs in steroidogenesis in mammals and insects, further accentuating the functional versatility of GSTs. We have provided an initial step for the development of potential treatments of steroidogenic disorders as well as tools for further investigation of activity of these GSTs in mammals.