Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM.
Elucidating the structural basis for differing enzyme inhibitor potency by cryo-EM.
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DOI：
10.1073/pnas.1708839115
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发表时间：
2018-02-20
影响因子：
11.1
通讯作者：
Muench SP
中科院分区：
文献类型：
--
作者：
Rawson S;Bisson C;Hurdiss DL;Fazal A;McPhillie MJ;Sedelnikova SE;Baker PJ;Rice DW;Muench SP
组氨酸生物合成是除草剂和抗菌剂的靶标，咪唑甘油磷酸酯酶（IGPD）是该途径中的关键酶。因此，IGPD是抑制剂设计计划的重点，有几种有效的除草剂正在开发中。有趣的是，与植物（拟南芥）IGPD相比，先导抑制剂对酵母（酵母）更有效。为了理解这种变化，我们已经通过电子显微镜确定了它们的结构，以揭示抑制剂效力差异背后的可能机制，其中酵母IGPD含有24个氨基酸的插入物，该插入物形成了一个延长的表面环，该表面环稳定了抑制剂结合环。这项研究提供了深入了解IGPD家族，并证明了使用电子显微镜方法研究抑制剂结合的能力。组氨酸的生物合成是植物和微生物的一个重要过程，使其成为开发除草剂和抗菌剂的一个有吸引力的目标。咪唑甘油-磷酸脱氢酶（Imidazoleglycerol-phosphate catalyase，IGPD）是这一途径中的关键酶，在酿酒酵母（Saccharomycescerevisiae，Sc_IGPD）和拟南芥（Arabidopsis thaliana，At_IGPD）中都有发现。作为抑制剂开发计划的一部分，植物酶一直是深入结构分析的重点，揭示了IGPD反应机制的细节，而酵母酶已被证明难以进行晶体学研究。IGPD的有效三唑-膦酸盐抑制剂的结构-活性关系已在两种同系物中确定，揭示了先导抑制剂（C348）对Sc_IGPD比At_IGPD更有效一个数量级;然而，这种差异的分子基础尚未建立。在这里，我们使用单粒子电子显微镜（EM）来研究At和Sc_IGPD同系物之间的结构差异，这可能会影响抑制剂效力的差异。在103 nm处得到的EM图谱足以从头构建蛋白质结构并鉴定抑制剂结合位点，这已经针对At_IGPD/C348复合物的晶体结构进行了验证。Sc_IGPD的结构表明，24个氨基酸的插入在酶表面上形成了一个延伸的环区域，该区域位于活性位点附近，与底物/抑制剂结合环形成相互作用，这可能影响抑制剂的效力。总体而言，这项研究提供了对IGPD家族的深入了解，并证明了使用EM方法研究抑制剂结合的能力。
Histidine biosynthesis is a target for herbicide and antibacterial agents, with imidazoleglycerol-phosphate dehydratase (IGPD) a key enzyme within this pathway. As a result, IGPD is the focus of inhibitor design programs, with several potent herbicides in development. Interestingly, the lead inhibitor is more potent against yeast (Saccharomyces) compared with plant (Arabidopsis) IGPD. To understand this change, we have determined their structure by electron microscopy to reveal a possible mechanism behind differences in inhibitor potency, with Saccharomyces IGPD containing a 24-amino acid insert that forms an extended surface loop that stabilizes an inhibitor binding loop. This study provides insights into the IGPD family and demonstrates the power of using an electron microcopy approach to study inhibitor binding. Histidine biosynthesis is an essential process in plants and microorganisms, making it an attractive target for the development of herbicides and antibacterial agents. Imidazoleglycerol-phosphate dehydratase (IGPD), a key enzyme within this pathway, has been biochemically characterized in both Saccharomyces cerevisiae (Sc_IGPD) and Arabidopsis thaliana (At_IGPD). The plant enzyme, having been the focus of in-depth structural analysis as part of an inhibitor development program, has revealed details about the reaction mechanism of IGPD, whereas the yeast enzyme has proven intractable to crystallography studies. The structure–activity relationship of potent triazole-phosphonate inhibitors of IGPD has been determined in both homologs, revealing that the lead inhibitor (C348) is an order of magnitude more potent against Sc_IGPD than At_IGPD; however, the molecular basis of this difference has not been established. Here we have used single-particle electron microscopy (EM) to study structural differences between the At and Sc_IGPD homologs, which could influence the difference in inhibitor potency. The resulting EM maps at ∼3 Å are sufficient to de novo build the protein structure and identify the inhibitor binding site, which has been validated against the crystal structure of the At_IGPD/C348 complex. The structure of Sc_IGPD reveals that a 24-amino acid insertion forms an extended loop region on the enzyme surface that lies adjacent to the active site, forming interactions with the substrate/inhibitor binding loop that may influence inhibitor potency. Overall, this study provides insights into the IGPD family and demonstrates the power of using an EM approach to study inhibitor binding.
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电子计数和束诱导运动校正可实现近原子分辨率的单粒子冷冻电镜。
影响因子：
48
作者：
Li, Xueming;Mooney, Paul;Zheng, Shawn;Booth, Christopher R.;Braunfeld, Michael B.;Gubbens, Sander;Agard, David A.;Cheng, Yifan
通讯作者：
Cheng, Yifan
影响因子：
14.8
作者：
Kelley LA;Mezulis S;Yates CM;Wass MN;Sternberg MJ
通讯作者：
Sternberg MJ
DOI：
10.1107/s2059798317004077
发表时间：
2017-06-01
期刊：
Acta crystallographica. Section D, Structural biology
影响因子：
--
作者：
Rawson S;McPhillie MJ;Johnson RM;Fishwick CWG;Muench SP
通讯作者：
Muench SP
DOI：
10.1016/j.str.2015.05.012
发表时间：
2015-07-07
期刊：
Structure (London, England : 1993)
影响因子：
--
作者：
Bisson C;Britton KL;Sedelnikova SE;Rodgers HF;Eadsforth TC;Viner RC;Hawkes TR;Baker PJ;Rice DW
通讯作者：
Rice DW
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH