Engineered Bacterial Progesterone 5β-Reductase: Tunable Substrate Preference and Synthesis of 5β-Dihydrosteroids

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The stereoselective hydrogenation of steroids at the 5β position is a crucial step in the synthesis of steroid drugs. Nevertheless, the suboptimal catalytic efficiency and poor heterologous expression levels of existing plant progesterone 5β-reductases (P5βRs) and animal-derived steroid 5β-reductases hinder their practical application. To expand the enzymatic synthesis pathway of 5β-dihydrosteroids, bacterial P5βRs were mined and their catalytic activities against progesterone and 8-oxogeranial were investigated. Similar to plant-derived PRISEs (progesterone 5β-reductase and/or iridoid synthase-like 1,4-enone reductases), bacterial P5βRs also exhibit divergent substrate preferences despite maintaining highly conserved protein sequences and structural architectures. Through integrated sequence-structure comparative analysis, a conformational switch controlling substrate selectivity was identified, which allows precise tuning of substrate preference in bacterial P5βR. Molecular dynamics (MD) simulation results indicate that the mutant M1 can open the cavity B within the substrate binding pocket, allowing the linear substrate 8-oxogeranial to stably bind. Furthermore, a substrate characteristics-oriented rational strategy was proposed to further enhance the catalytic activity of bacterial P5βR toward steroids. The progesterone catalytic efficiency of the optimal mutant Lp P5βR-M5 was increased by more than 700-fold compared with the wild type. The gram-level preparation of 5β-pregnane-3,20-dione showed that Lp P5βR-M5 could almost completely convert 28 g/L progesterone within 2 h (conv. >98%), and the space-time yield (STY) was as high as 330 g/L·d. In addition, the structural basis for the improved catalytic activity of Lp P5βR-M5 was explored employing MD simulation and caver analysis. This study not only elucidates the structure–function relationship of bacterial P5βR but also pioneers an environmentally friendly biocatalytic pathway for 5β-dihydrosteroid synthesis.