Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, Innovation Academy of South China Sea Ecology and Environmental Engineering, and South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, China.
Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, Innovation Academy of South China Sea Ecology and Environmental Engineering, and South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; University of the Chinese Academy of Sciences, Beijing, China.
J Biol Chem. 2020 Dec 11;295(50):16987-16997. doi: 10.1074/jbc.RA120.015563. Epub 2020 Oct 1.
Epoxide hydrolases (EHs) have been characterized and engineered as biocatalysts that convert epoxides to valuable chiral vicinal diol precursors of drugs and bioactive compounds. Nonetheless, the regioselectivity control of the epoxide ring opening by EHs remains challenging. Alp1U is an α/β-fold EH that exhibits poor regioselectivity in the epoxide hydrolysis of fluostatin C (compound 1) and produces a pair of stereoisomers. Herein, we established the absolute configuration of the two stereoisomeric products and determined the crystal structure of Alp1U. A Trp-186/Trp-187/Tyr-247 oxirane oxygen hole was identified in Alp1U that replaced the canonical Tyr/Tyr pair in α/β-EHs. Mutation of residues in the atypical oxirane oxygen hole of Alp1U improved the regioselectivity for epoxide hydrolysis on 1. The single site Y247F mutation led to highly regioselective (98%) attack at C-3 of 1, whereas the double mutation W187F/Y247F resulted in regioselective (94%) nucleophilic attack at C-2. Furthermore, single-crystal X-ray structures of the two regioselective Alp1U variants in complex with 1 were determined. These findings allowed insights into the reaction details of Alp1U and provided a new approach for engineering regioselective epoxide hydrolases.
环氧化物水解酶（EHs）已被鉴定和设计为生物催化剂，可将环氧化物转化为有价值的手性邻二醇前体药物和生物活性化合物。尽管如此，EHs 对环氧开环的区域选择性控制仍然具有挑战性。Alp1U 是一种 α/β 折叠 EH，在氟司他汀 C（化合物 1）的环氧化物水解中表现出较差的区域选择性，产生一对立体异构体。在此，我们确定了这两种立体异构体产物的绝对构型，并确定了 Alp1U 的晶体结构。在 Alp1U 中鉴定出一个 Trp-186/Trp-187/Tyr-247 环氧化物氧穴，取代了 α/β-EHs 中的典型 Tyr/Tyr 对。Alp1U 中非典型环氧化物氧穴中残基的突变提高了 1 的环氧化物水解的区域选择性。单个位置 Y247F 突变导致 1 的 C-3 高度区域选择性（98%）进攻，而双突变 W187F/Y247F 导致 C-2 的区域选择性（94%）亲核进攻。此外，还确定了与 1 结合的两种区域选择性 Alp1U 变体的单晶 X 射线结构。这些发现使我们深入了解了 Alp1U 的反应细节，并为工程化区域选择性环氧化物水解酶提供了一种新方法。
