State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, PR China.
State Key Laboratory of Physical Chemistry of Solid Surfaces and Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 360015, PR China.
Biochem Biophys Res Commun. 2020 Aug 20;529(2):156-161. doi: 10.1016/j.bbrc.2020.05.199. Epub 2020 Jun 22.
Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O phenolate anion then attacks the nearby C5, and triggers intramolecular S2' syn displacement with the elimination of O-acetyl group. Moreover, the latter S2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.
海洛因合成酶 2（THS2）可以将（7S）-萨尔塔定醇 7-O-乙酸转化为蒂巴因，催化罂粟中蒂巴因生物合成的最后一步。本文报道了 THS2 及其与蒂巴因复合物的晶体结构，揭示了底物结合口袋中的相互作用网络。随后进行了对接和 QM/MM 研究，以进一步探讨 THS2 的催化机制。我们的结果表明，在 H89 的协助下，T105 可能从底物中提取 C4-OH 的质子。由此产生的 C4-O 酚盐阴离子然后攻击附近的 C5，并引发分子内 S2' 顺式取代，同时消除 O-乙酰基。此外，后一步 S2' 反应是整个酶反应的速率决定步骤，总能量势垒为 18.8 kcal/mol。这些发现对于理解蒂巴因生物合成的作用机制至关重要，并将指导通过代谢工程增强鸦片生物碱生产的酶工程。
