Burris-Hiday Sarah D, Scott Emily E
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan, USA; Departments of Pharmacology and Biological Chemistry and the Programs in Chemical Biology and Biophysics, University of Michigan, Ann Arbor, Michigan, USA.
J Biol Chem. 2023 Sep;299(9):105112. doi: 10.1016/j.jbc.2023.105112. Epub 2023 Jul 28.
NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.
烟酰胺腺嘌呤二核苷酸磷酸-细胞色素P450还原酶提供血红素加氧酶、角鲨烯单加氧酶、脂肪酸去饱和酶以及48种人细胞色素P450酶所需的电子。虽然支持还原酶分子内电子转移的构象变化已得到充分阐明，但与这些靶标的分子间相互作用却了解甚少，部分原因是它们的瞬时结合。在此，负责与靶标相互作用的还原酶黄素单核苷酸（FMN）结构域与三种药物代谢型和两种类固醇生成型细胞色素P450酶的N端融合，以增加相互作用的概率。对这些人工融合酶结合各自底物和抑制剂的能力以及在氢过氧化异丙苯支持下进行催化的能力进行了分析。与分离的P450酶的比较表明，即使是氧化的FMN结构域也会对P450功能产生大量且多样的影响。FMN结构域可能会增加、降低或不影响总配体结合和/或解离常数，这取决于P450酶和配体。例如，FMN结构域融合对抑制剂酮康唑与CYP17A1的结合没有影响，但却显著改变了同一化合物与CYP21A2的结合。FMN结构域与CYP21A2的融合导致了不同的效应，这取决于配体是17α-羟孕酮还是酮康唑。在稳态动力学方面也观察到了类似的酶特异性效应。这些观察结果最符合FMN结构域与近端P450表面相互作用，以别构方式影响P450配体结合和代谢，而与电子传递无关。对不同P450酶以及对同一P450与不同配体产生的各种效应表明，P450活性位点与其近端还原酶结合表面之间存在复杂且不同的别构通讯。
