X-ray crystal structures of D100E trichodiene synthase and its pyrophosphate complex reveal the basis for terpene product diversity
X-ray crystal structures of D100E trichodiene synthase and its pyrophosphate complex reveal the basis for terpene product diversity
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DOI：
10.1021/bi011960g
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发表时间：
2002-02-12
期刊：
影响因子：
2.9
通讯作者：
Christianson, DW
中科院分区：
文献类型：
--
作者：
Rynkiewicz, MJ;Cane, DE;Christianson, DW
报道了D100 E β-二烯合酶的2.4埃分辨X射线晶体结构及其与无机焦磷酸盐复合物的2.6埃分辨结构。在所谓的“富含谷氨酸”基序中的D100 E氨基酸取代不会导致酶的整体结构发生大的变化。然而，在焦磷酸盐复合物中，焦磷酸盐在活性位点的口处与两个Mg 2+离子配位，而不会引起酶结构的大的变化。这与野生型酶中的焦磷酸盐结合形成对比，其中焦磷酸盐与三个Mg 2+离子配位并引发显著的构象变化，该构象变化关闭活性位点的口并优化酶-底物复合物中的堆积密度。在D100 E β-二烯合酶的活性位点封闭的衰减妥协酶底物的堆积密度，并赋予额外的空间和构象自由度的基板和碳阳离子中间体，这反过来又导致在形成五个替代的倍半萜产品除了β-二烯。因此，生物学中萜烯环化酶的多样性可能部分是通过氨基酸取代而进化的，氨基酸取代可以微调依赖于金属-二磷酸络合的结构变化，金属-二磷酸络合控制活性位点模板和酶-底物堆积密度的形成。
The 2.4 Angstrom resolution X-ray crystal structure of D100E trichodiene synthase and the 2.6 Angstrom resolution structure of its complex with inorganic pyrophosphate are reported. The D100E amino acid substitution in the so-called "aspartate-rich" motif does not result in large changes to the overall structure of the enzyme. In the pyrophosphate complex, however, pyrophosphate coordinates two Mg2+ ions at the mouth of the active site without causing large changes in the structure of the enzyme. This contrasts with pyrophosphate binding in the wild-type enzyme, where pyrophosphate coordinates three Mg2+ ions and triggers a significant conformational change that closes the mouth of the active site and optimizes packing density in the enzyme-substrate complex. The attenuation of active site closure in D100E trichodiene synthase compromises enzyme-substrate packing density and confers additional spatial and conformational degrees of freedom on the substrate and carbocation intermediates, which in turn results in the formation of five alternate sesquiterpene products in addition to trichodiene. By extension, then, the diversity of terpene cyclases in biology may have evolved in part by amino acid substitutions that fine-tune structural changes dependent on metal-diphosphate complexation that govern the formation of the active site template and enzyme-substrate packing density.