Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.
Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.
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DOI：
10.1016/j.jbc.2021.100645
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Zhang H
中科院分区：
文献类型：
--
作者：
Cheng S;Bo Z;Hollenberg P;Osawa Y;Zhang H
细胞色素P450（CYP）与NADPH-细胞色素P450氧化还原酶（POR）的相互作用是外源性物质代谢所必需的，膜脂促进了这种相互作用。各种膜模拟物，如磷脂脂质体和纳米盘，已被用于模拟膜以形成催化活性的P0R：P0R复合物。然而，这些相互作用的确切机制尚不清楚，因为缺乏膜中全长哺乳动物P0R：POR复合物的结构信息。在此，我们报告使用的amphipols（APols），形成一个功能齐全，可溶性，均匀的制备全长的pH-POR复合物服从生化和结构研究。将CYP2B4和POR结合到APols中，产生了化学计量比为1：1的CYP2B4：POR复合物，其在甲基苄非他明的脱甲基中具有完全功能，转换速率为37.7 ± 2.2 min − 1，偶联效率为40%。有趣的是，通过多角度光散射测定的稳定复合物的分子量（Mw）为338 ± 22 kDa，表明2CYP 2B4：2POR的四聚体复合物包埋在一个APol纳米颗粒中。此外，负染色电子显微镜（EM）验证了复合物的均匀性，并允许我们生成一个三维EM图和模型与在溶液中观察到的四聚体一致。这是第一次报告的全长哺乳动物的ESTA：POR复合物的传输EM不仅揭示了架构，有利于电子转移，但也突出了潜在的使用APols的功能性ESTA复合物的生化和结构研究与氧化还原合作伙伴。
Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min−1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.
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影响因子：
48
作者：
Kastner, Berthold;Fischer, Niels;Stark, Holger
通讯作者：
Stark, Holger
DOI：
10.1073/pnas.062565599
发表时间：
2002-05-14
影响因子：
11.1
作者：
Bayburt, TH;Sligar, SG
通讯作者：
Sligar, SG
影响因子：
4.9
作者：
Mahajan, Mukesh;Ravula, Thirupathi;Ramamoorthy, Ayyalusamy
通讯作者：
Ramamoorthy, Ayyalusamy
DOI：
10.1016/0006-291x(79)91238-5
发表时间：
1979-01-01
影响因子：
3.1
作者：
BLACK, SD;FRENCH, JS;COON, MJ
通讯作者：
COON, MJ
DOI：
10.1002/anie.201802210
发表时间：
2018-07-09
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Prade E;Mahajan M;Im SC;Zhang M;Gentry KA;Anantharamaiah GM;Waskell L;Ramamoorthy A
通讯作者：
Ramamoorthy A