The United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1194, Japan; Center for One Medicine Innovative Translational Research, Gifu University, Gifu 501-1193, Japan.
Forensic Science Laboratory, Gifu Prefectural Police Headquarters, Gifu 500-8501, Japan.
J Steroid Biochem Mol Biol. 2024 Oct;243:106574. doi: 10.1016/j.jsbmb.2024.106574. Epub 2024 Jun 28.
Porcine carbonyl reductases (pCBR1 and pCBR-N1) and aldo-keto reductases (pAKR1C1 and pAKR1C4) exhibit hydroxysteroid dehydrogenase (HSD) activity. However, their roles in the metabolism of porcine-specific androgens (19-nortestosterone and epiandrosterone), 11-oxygenated androgens, neurosteroids, and corticosteroids remain unclear. Here, we compared the steroid specificity of the four recombinant enzymes by kinetic and product analyses. In C/C-steroids,11-keto- and 11β-hydroxy-5α-androstane-3,17-diones were reduced by all the enzymes, whereas 5α-dihydronandrolone (19-nortestosterone metabolite) and 11-ketodihydrotestosterone were reduced by pCBR1, pCBR-N1, and pAKR1C1, of which pCBR1 exhibited the lowest (submicromolar) K values. Product analysis showed that pCBR1 and pCBR-N1 function as 3α/β-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3β-HSD and 3α-HSD, respectively). Additionally, 17β-HSD activity was observed in pCBR1 and pCBR-N1 (toward epiandrosterone and its 11-oxygenated derivatives) and in pAKR1C1 (toward androsterone, 4-androstene-3,17-dione and their 11-oxygenated derivatives). The four enzymes also showed different substrate specificity for 3-keto-5α/β-dihydro-C-steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5β-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/β-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/β-dihydro-metabolites of cortisol, 11-deoxycortisol, cortisone, and corticosterone. pCBR1 exhibited lower K values (0.3-2.9 μM) for the 3-keto-C-steroids than pCBR-N1 (K=10-36 μM). The reduced products of the 3-keto-C-steroids by pCBR1 and pCBR-N1 were their 3α-hydroxy-metabolites. Finally, we found that human CBR1 has similar substrate specificity for the C/C/C-steroids to pCBR-N1. Based on these results, it was concluded that porcine and human CBRs can be involved in the metabolism of the aforementioned steroids as 3α/β,17β-HSDs.
猪的羰基还原酶 (pCBR1 和 pCBR-N1) 和醛酮还原酶 (pAKR1C1 和 pAKR1C4) 表现出羟甾体脱氢酶 (HSD) 活性。然而，它们在代谢猪特有的雄激素 (19-去甲睾酮和表雄酮)、11-氧化雄激素、神经甾体和皮质甾类中的作用仍不清楚。在这里，我们通过动力学和产物分析比较了这四种重组酶的甾体特异性。在 C/C-甾体中，所有酶都能还原 11-酮基和 11β-羟基-5α-雄烷-3,17-二酮，而 5α-二氢雄酮 (19-去甲睾酮代谢物) 和 11-酮基二氢睾酮则被 pCBR1、pCBR-N1 和 pAKR1C1 还原，其中 pCBR1 的 K 值最低 (亚微摩尔)。产物分析表明，pCBR1 和 pCBR-N1 作为 3α/β-HSD 发挥作用，而 pAKR1C1 和 pAKR1C4 则分别作为 3β-HSD 和 3α-HSD 发挥作用。此外，pCBR1 和 pCBR-N1 对表雄酮及其 11-氧化衍生物) 和 pAKR1C1 (对雄酮、4-雄烯-3,17-二酮及其 11-氧化衍生物) 表现出 17β-HSD 活性。四种酶对 3-酮-5α/β-二氢-C-甾体也表现出不同的底物特异性，包括 GABA 能神经甾体前体和皮质甾类代谢物。所有酶都能还原 5β-二氢孕酮，而 pCBR1 能还原 5α-二氢孕酮，pCBR1 和 pCBR-N1 能还原 5α/β-二氢去氧皮质甾酮。这两种 pCBR 还能还原皮质醇、11-脱氧皮质醇、皮质酮和皮质醇的 5α/β-二氢代谢物。pCBR1 对 3-酮-C-甾体的 K 值 (0.3-2.9 μM) 低于 pCBR-N1 (K=10-36 μM)。pCBR1 和 pCBR-N1 还原的 3-酮-C-甾体的产物是它们的 3α-羟基代谢物。最后，我们发现人 CBR1 对 C/C/C-甾体的底物特异性与 pCBR-N1 相似。基于这些结果，可以得出结论，猪和人 CBR 可以作为 3α/β、17β-HSD 参与上述甾体的代谢。
