Molecular recognition of the substrate diphosphate group governs product diversity in trichodiene synthase mutants.
Molecular recognition of the substrate diphosphate group governs product diversity in trichodiene synthase mutants.
复制标题
DOI：
10.1021/bi050059o
复制
发表时间：
2005-03
期刊：
影响因子：
2.9
通讯作者：
L. Vedula;M. Rynkiewicz;H. Pyun;R. Coates;D. Cane;D. Christianson
中科院分区：
文献类型：
--
作者：
L. Vedula;M. Rynkiewicz;H. Pyun;R. Coates;D. Cane;D. Christianson
报道了Y305F毛状二烯合成酶及其与副产物无机焦磷酸(PP(I))的络合物，以及Y305F和D100E毛状二烯合成酶与PP(I)和双苯并碳阳离子的氮杂类似物形成的三元络合物的X射线晶体结构。与野生型酶相比，碱性D(302)RRYR基序中的Y305F取代对未复合酶及其与PP(I)的络合物的整体结构没有太大的影响。然而，Y305F-PP(I)氢键的损失似乎被R304侧链位置的非常轻微的移动所补偿。推测的双酚基碳正离子R-氮杂双异丁烯的构象和取向似乎并不模仿实际的碳正离子中间体的构象和取向，这表明R-和S-氮杂双异丁烯的强烈抑制更多地来自于与PP(I)的有利静电相互作用，而不是与反应中间体的任何特殊相似之处。Y305F和D100E突变导致更大的封闭活性中心体积，这些突变似乎赋予配体结合构象和取向更大的可变性，从而导致异常环化产物的形成。由于R-氮杂异构体与Y305F和D100E三环烯合成酶的结合构象和取向不对应于产物形成所需的结合构象，而且不同底物和碳正离子类似物与其他环酶如5-表马兜铃烯合成酶和龙脑基二磷酸合成酶的结合构象和取向通常不对应于催化生成的络合物，因此我们得出结论，萜烯环化反应中瞬时碳正离子中间体的形成一般是受动力学而不是热力学控制的。
The X-ray crystal structures of Y305F trichodiene synthase and its complex with coproduct inorganic pyrophosphate (PP(i)) and of Y305F and D100E trichodiene synthases in ternary complexes with PP(i) and aza analogues of the bisabolyl carbocation intermediate are reported. The Y305F substitution in the basic D(302)RRYR motif does not cause large changes in the overall structure in comparison with the wild-type enzyme in either the uncomplexed enzyme or its complex with PP(i). However, the loss of the Y305F-PP(i) hydrogen bond appears to be compensated by a very slight shift in the position of the side chain of R304. The putative bisabolyl carbocation mimic, R-azabisabolene, binds in a conformation and orientation that does not appear to mimic that of the actual carbocation intermediate, suggesting that the avid inhibition by R- and S-azabisabolenes arises more from favorable electrostatic interactions with PP(i) rather than any special resemblance to a reaction intermediate. Greater enclosed active-site volumes result from the Y305F and D100E mutations that appear to confer greater variability in ligand-binding conformations and orientations, which results in the formation of aberrant cyclization products. Because the binding conformations and orientations of R-azabisabolene to Y305F and D100E trichodiene synthases do not correspond to binding conformations required for product formation and because the binding conformations and orientations of diverse substrate and carbocation analogues to other cyclases such as 5-epi-aristolochene synthase and bornyl diphosphate synthase generally do not correspond to catalytically productive complexes, we conclude that the formation of transient carbocation intermediates in terpene cyclization reactions is generally under kinetic rather than thermodynamic control.