Structural characterization and Kemp eliminase activity of the Mycobacterium smegmatis Ketosteroid Isomerase
Structural characterization and Kemp eliminase activity of the Mycobacterium smegmatis Ketosteroid Isomerase
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耻垢分枝杆菌酮类固醇异构酶的结构特征和 Kemp 消除酶活性
DOI：
10.1016/j.bbrc.2021.05.007
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发表时间：
2021
影响因子：
3.1
通讯作者：
Mark Bartlam
中科院分区：
文献类型：
--
作者：
Yakun Liang;Weiping Li;Han Liang;Xiaorui Lou;Ruihua Liu;Qionglin Zhang;Mark Bartlam
肯普消除反应涉及苯并恶唑及其衍生物在天然酶或化学催化剂作用下的开环反应，自发现以来一直引起研究人员的兴趣。由于这种反应并不存在于所有目前已知的代谢途径，肯普消除酶的计算设计提供了有价值的见解酶催化的原则。然而，发现天然存在的混杂酶ydbC、xapA和甾酮异构酶也可以催化肯普消除。本文报道了耻垢分枝杆菌MC 2 155的甾酮异构酶（KSI）的晶体结构。MsKSI晶体属于P212121空间群，在一个不对称单元中有两个分子，超离心数据证实它在溶液中形成一个稳定的二聚体，与1.9 μ m分辨率的结构一致。我们的试验证实MsKSI加速肯普消除5-硝基苯并恶唑（5 NBI）的最佳pH值为5.5。MsKSI-5 NBI复合物的2.35 μ m分辨率的晶体结构表明，底物5 NBI结合在由疏水残基组成的酶的活性口袋中。此外，Glu 127残基被认为在质子转移和断开弱O-N键以打开五元环中作为一般碱发挥重要作用。本工作为探索以天然酶为骨架对MsKSI进行人工修饰奠定了基础。
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 enzymesydbC,xapA and ketosteroid isomerase also can catalyze Kemp elimination. Here, we report the crystal structure of ketosteroid isomerase (KSI) fromMycobacterium smegmatisMC2 155.MsKSI crystallizes in the P212121space 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 thatMsKSI accelerates the Kemp elimination of 5-nitrobenzoxazole (5NBI) with an optimal pH of 5.5. A 2.35 Å resolution crystal structure of theMsKSI-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 ofMsKSI using the natural enzyme as the backbone.