Steric enforcement of cis-epoxide formation in the radical C-O-coupling reaction by which (S)-2-hydroxypropylphosphonate epoxidase (HppE) produces Fosfomycin.
Steric enforcement of cis-epoxide formation in the radical C-O-coupling reaction by which (S)-2-hydroxypropylphosphonate epoxidase (HppE) produces Fosfomycin.
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DOI：
10.1021/jacs.9b10974
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发表时间：
2019-11
影响因子：
15
通讯作者：
Shengbin Zhou;Juan Pan;Katherine M Davis;Irene Schaperdoth;Bo Wang;A. Boal;C. Krebs;J. Bollinger-J.-Bolli
中科院分区：
文献类型：
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作者：
Shengbin Zhou;Juan Pan;Katherine M Davis;Irene Schaperdoth;Bo Wang;A. Boal;C. Krebs;J. Bollinger-J.-Bolli
(S)-2-羟丙基膦酸酯 [(S)-2-HPP, 1] 环氧酶 (HppE) 在其非血红素铁辅助因子处还原 H2O2，以安装抗生素磷霉素的环氧乙烷“弹头”。 1 的 C1 pro-R 氢被其 C2 氧净取代，并在 C1 处构型反转，产生药物的顺式环氧化物 [(1R,2S)-环氧丙基膦酸 (cis-Fos, 2)]。在这里，我们表明，HppE 通过自由基偶联机制的空间引导，对 C1 反转和顺式环氧化物形成具有约 95% 的选择性。已发表的HppE•FeII•1 和HppE•ZnII•2 复合物的结构揭示了底物和产物的C3 的不同口袋，并确定了四个疏水残基——Leu120、Leu144、Phe182 和Leu193——靠近其中一个复合物中的C3。用体积更大的 Phe 替换底物 C3 口袋中的 Leu193 可增强立体选择性（顺式：反式 ~ 99:1），而产物 C3 口袋中的 Leu120Phe 替代会减弱立体选择性（~ 82:18）。随着底物 C3 口袋中体积还原性 Phe182Ala 取代 (~ 13:87)、C3 三氟化 (~ 23:77) 或两者 (~ 1:99)，C1 构型的保留和反式环氧化物的形成成为主导。 C3 三氟化的影响被 Leu193Phe (~ 56:44) 和 Leu144Phe/Leu193Phe (~ 90:10) 变体中更受限制的底物 C3 口袋抵消。 HppE 环氧化 C3、C1 或两者上带有卤素的底物类似物的能力与已发表的通过 C1 碳阳离子极性环化的假设不一致。相反，特定的酶-底物接触会驱动 C1 自由基的反转（正如最近的一项计算研究中提出的那样），从而通过类自由基 C-O 偶联直接形成更有效的抗菌顺式环氧化物。
(S)-2-hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its non-heme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves ~ 95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE•FeII•1 and HppE•ZnII•2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues - Leu120, Leu144, Phe182, and Leu193 - close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans ~ 99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (~ 82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (~ 13:87), trifluorination of C3 (~ 23:77), or both (~ 1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (~ 56:44) and Leu144Phe/Leu193Phe (~ 90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical - as proposed in a recent computational study - to direct formation of the more potently antibacterial cis epoxide by radicaloid C-O coupling.