Laboratory of Biophysical Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands.
J Biol Chem. 2012 Sep 7;287(37):30975-83. doi: 10.1074/jbc.M112.374306. Epub 2012 Jul 24.
3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.
3-酮类固醇 Δ4-(5α)-脱氢酶（Δ4-(5α)-KSTDs）是一种酶，它在 3-酮-(5α)-类固醇的 C4 和 C5 原子之间引入双键。在这里，我们表明来自节杆菌 RHA1 的 ro05698 基因编码具有 Δ4-(5α)-KSTD 活性的黄素蛋白。该酶的 1.6 Å 分辨率晶体结构揭示了 FAD 辅因子异咯嗪环系统附近口袋中的三个保守残基（Tyr-319、Tyr-466 和 Ser-468）。这些残基的定点突变证实它们对催化活性是绝对必需的。与结合产物 4-雄烯-3,17-二酮的晶体结构表明，Ser-468 处于可以作为从底物的 C4 原子上提取 4β-质子的碱基的位置。Ser-468 由 Tyr-319 辅助，Tyr-319 可能参与将质子转移到溶剂中。Tyr-466 与底物的 C3 氧原子处于氢键距离，可以稳定反应过程中发生的酮-烯醇中间物。最后，FAD N5 原子的位置能够将底物的 5α-氢作为氢化物离子进行提取。这些特征完全解释了 Δ4-(5α)-KSTDs 催化的反应。
