Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
Biochemistry. 2010 Mar 2;49(8):1787-97. doi: 10.1021/bi902088z.
The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 A resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg(2+) ions, inorganic pyrophosphate (PP(i)), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 A resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation-pi interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 A resolution crystal structure of F198A EIZS in a complex with three Mg(2+) ions, PP(i), and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg(2+) ions has been determined at 1.90 A resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.
重组表异泽兰烯合酶（EIZS）的 X 射线晶体结构，来自于变铅青链霉菌 A3(2) 的一种倍半萜环化酶，已在 1.60 A 分辨率下确定。具体而言，野生型 EIZS 的结构为其与三个 Mg(2+) 离子、无机焦磷酸（PP(i)）和苄基三乙基铵阳离子（BTAC）形成的封闭构象。此外，D99N EIZS 的结构已在 1.90 A 分辨率下确定为开放的、无配体的构象。这两种结构的比较提供了细菌萜烯环化酶中底物结合和催化所需构象变化的第一个视图。此外，BTAC 的结合相互作用可能模拟催化过程中碳正离子中间体。因此，F95、F96 和 F198 的芳环似乎很好地定向，通过阳离子-π 相互作用稳定环化级联中的碳正离子中间体。酶活性位点中芳香残基的突变导致产生替代的倍半萜烯产物阵列，这是由于碳正离子中间体的稳定模式以及法呢基二磷酸环化的模板发生改变。因此，F198A EIZS 与三个 Mg(2+) 离子、PP(i) 和 BTAC 形成的复合物的 1.64 A 分辨率晶体结构揭示了 BTAC 的替代结合取向；碳正离子中间体的替代结合取向可能导致形成替代产物。最后，野生型 EIZS 与四个 Hg(2+) 离子形成的复合物的 X 射线晶体结构已在 1.90 A 分辨率下确定，表明金属结合触发了 G 螺旋的显著构象变化，以覆盖活性位点。
