3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy
3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy
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通过顺磁 NMR 波谱法测定不稳定瞬时酶中间体的 3D 结构。
DOI：
10.1002/anie.201606223
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发表时间：
2016-10-24
影响因子：
16.6
通讯作者：
Su, Xun-Cheng
中科院分区：
文献类型：
--
作者：
Chen, Jia-Liang;Wang, Xiao;Su, Xun-Cheng
酶催化依赖于构象可塑性，但瞬态中间体的结构信息很难获得。我们表明，一个不稳定的，低丰度的酶中间体的三维（3D）结构，可以通过核磁共振（NMR）光谱。该方法被证明用于金黄色葡萄球菌分选酶A（SrtA），这是一种既定的药物靶标和生物技术试剂。 SrtA是将底物肽的酰胺键转化为硫酯的转肽酶。通过测量由不与活性位点残基Cys184反应的位点特异性半胱氨酸反应性顺磁性标签产生的假接触位移（PCS），收集足够数量的限制以确定SrtA的不稳定硫酯中间体的3D结构，所述SrtA在非平衡条件下仅作为次要物质存在。3D结构揭示了保护硫酯中间体免受水解的结构变化。
Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three-dimensional (3D) structure of an unstable, low-abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site-specific cysteine-reactive paramagnetic tag that does not react with the active-site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non-equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.