State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science and College of Life Sciences, Nankai University, Tianjin, 300071, China.
State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Protein Science and College of Life Sciences, Nankai University, Tianjin, 300071, China.
Biochem Biophys Res Commun. 2021 Jun 30;560:159-164. doi: 10.1016/j.bbrc.2021.05.007. Epub 2021 May 13.
The Kemp elimination reaction, involving the ring-opening of benzoxazole and its derivatives under the action of natural enzymes or chemical catalysts, has been of interest to researchers since its discovery. Because this reaction does not exist in all currently known metabolic pathways, the computational design of Kemp eliminases has provided valuable insights into principles of enzymatic catalysis. However, it was discovered that the naturally occurring promiscuous enzymes ydbC, xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) from Mycobacterium smegmatis MC2 155. MsKSI crystallizes in the P222 space group with two molecules in an asymmetric unit, and ultracentrifugation data confirms that it forms a stable dimer in solution, consistent with the 1.9 Å-resolution structure. Our assays confirm that MsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of the MsKSI-5NBI complex reveals that the substrate 5NBI is bound in the active pocket of the enzyme composed of hydrophobic residues. In addition, the Glu127 residue is proposed to play an important role as a general base in proton transfer and breaking weak O-N bonds to open the five-membered ring. This work provides a starting point for exploring the artificial modification of MsKSI using the natural enzyme as the backbone.
肯普消除反应是指苯并恶唑及其衍生物在天然酶或化学催化剂的作用下开环，自发现以来一直受到研究人员的关注。由于这种反应不存在于所有已知的代谢途径中，因此肯普消除酶的计算设计为酶催化的原理提供了有价值的见解。然而，人们发现天然存在的混杂酶 ydbC、xapA 和酮固醇异构酶也可以催化肯普消除。在这里，我们报告了分枝杆菌 MC2 155 酮固醇异构酶（KSI）的晶体结构。MsKSI 结晶在 P222 空间群中，每个不对称单元包含两个分子，超速离心数据证实它在溶液中形成稳定的二聚体，与 1.9 Å分辨率的结构一致。我们的测定证实 MsKSI 能以最佳 pH 值 5.5 加速 5-硝基苯并恶唑（5NBI）的肯普消除。MsKSI-5NBI 复合物的 2.35 Å 分辨率晶体结构表明，底物 5NBI 结合在由疏水残基组成的酶的活性口袋中。此外，推测 Glu127 残基在质子转移和打破弱 O-N 键以打开五元环中起重要作用作为一个通用的基地。这项工作为使用天然酶作为骨架探索 MsKSI 的人工修饰提供了一个起点。
