Identification of gut bacteria reductases that biotransform steroid hormones

The metabolism of steroid hormones by the gut microbiome is increasingly recognized as a key factor in human health; however, the specific enzymes mediating these transformations remain largely unidentified. In this study, we identify Δ⁴-3-ketosteroid 5β-reductase, 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase, and Δ⁶-3-ketosteroid reductase enzyme families encoded by common human gut bacteria. Through phylogenetic reconstruction and mutagenesis, we show that 5β-reductase evolved to specialize in converting both natural and synthetic 3-ketosteroid hormones into their 5β-reduced derivatives, while Δ⁶-3-ketosteroid reductase adapted to produce Δ⁶-reduced derivatives. We also find that the novel 3β-hydroxysteroid dehydrogenase/Δ^5-4 isomerase is fused with 5β-reductase in multiple species, streamlining the conversion of pregnenolone, a 3β-hydroxy-5-ene and steroid hormone precursor, into epipregnanolone. Through metagenomic analysis, we reveal that these enzymes are prevalent in healthy populations and enriched in females compared to males. These findings lay the groundwork for mechanistic investigations into how microbial steroid metabolism modulates host hormonal physiology.