Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland.
The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland.
Int J Mol Sci. 2022 Nov 24;23(23):14660. doi: 10.3390/ijms232314660.
Bacteria and fungi that are able to metabolize steroids express 3-ketosteroid-Δ-dehydrogenases (KstDs). KstDs such as AcmB form Chol-1 catalyze the enantioselective 1α,2β-dehydrogenation of steroids to their desaturated analogues, e.g., the formation of 1,4-androstadiene-3,17-dione (ADD) from 4-androsten-3,17-dione (AD). The reaction catalyzed by KstD can be reversed if the appropriate electron donor, such as benzyl viologen radical cation, is present. Furthermore, KstDs can also catalyze transhydrogenation, which is the transfer of H atoms between 3-ketosteroids and 1-dehydrosteroids. In this paper, we showed that AcmB exhibits lower pH optima for hydrogenation and dehydrogenation by 3.5-4 pH units than those observed for KstD from We confirmed the enantiospecificity of 1α,2β-hydrogenation and 1α,2β-transhydrogenation catalyzed by AcmB and showed that, under acidic pH conditions, deuterons are introduced not only at 2β but also at the 1α position. We observed a higher degree of H/D exchange at Y363, which activates the C2-H bond, compared to that at FAD, which is responsible for redox at the C1 position. Furthermore, for the first time, we observed the introduction of the third deuteron into the steroid core. This effect was explained through a combination of LC-MS experiments and QM:MM modelling, and we attribute it to a decrease in the enantioselectivity of C2-H activation upon the deuteration of the 2β position. The increase in the activation barrier resulting from isotopic substitution increases the chance of the formation of d-substituted 3-ketosteroids. Finally, we demonstrate a method for the synthesis of 3-ketosteroids chirally deuterated at 1α,2β positions, obtaining 1α,2β-d-4-androsten-3,17-dione with a 51% yield (8.61 mg).
能够代谢类固醇的细菌和真菌表达 3-酮类固醇-Δ-脱氢酶（KstD）。像 AcmB 这样的 KstD 形成 Chol-1，可催化类固醇的对映选择性 1α，2β-脱氢，生成其不饱和类似物，例如 4-雄烯-3，17-二酮（AD）转化为 1，4-雄二烯-3，17-二酮（ADD）。如果存在合适的电子供体，如苄基紫精自由基阳离子，则可以逆转由 KstD 催化的反应。此外，KstD 还可以催化氢转移，即 3-酮固醇和 1-脱氢固醇之间的 H 原子转移。在本文中，我们表明 AcmB 对 3-酮固醇的加氢和脱氢的 pH 最优值比从 We 鉴定的 KstD 低 3.5-4 pH 单位。我们证实了 AcmB 催化的 1α，2β-加氢和 1α，2β-氢转移的对映体特异性，并表明在酸性 pH 条件下，不仅在 2β位，而且在 1α位引入了氘原子。与负责 C1 位氧化还原的 FAD 相比，我们观察到在激活 C2-H 键的 Y363 处有更高程度的 H/D 交换。此外，我们首次观察到第三个氘原子引入类固醇核心。通过 LC-MS 实验和 QM:MM 建模的结合，我们解释了这种效应，并将其归因于 2β 位氘化导致 C2-H 活化的对映选择性降低。由于同位素取代而增加的活化能垒增加了形成 d-取代 3-酮固醇的机会。最后，我们展示了一种在 1α，2β 位手性氘化 3-酮固醇的合成方法，以 51%的收率（8.61mg）获得 1α，2β-d-4-雄烯-3，17-二酮。
