Jerabek Petr, Martinek Vaclav, Stiborova Marie
Department of Biochemistry, Charles University, Prague, Czech Republic.
Neuro Endocrinol Lett. 2012;33 Suppl 3:25-32.
The herbal drug aristolochic acid (AA) derived from Aristolochia species has been shown to be the cause of aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and their urothelial malignancies. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from nephropathy and tumor development. One cause for these different responses may be individual differences in the activities of the enzymes catalyzing the biotransformation of AA. Thus, the identification of enzymes principally involved in the metabolism of AAI, the major toxic component of AA, and detailed knowledge of their catalytic specificities is of major importance. Human cytochrome P450 (CYP) 1A1 and 1A2 enzymes were found to be responsible for the AAI reductive activation to form AAI-DNA adducts, while its structurally related analogue, CYP1B1 is almost without such activity. However, knowledge of the differences in mechanistic details of CYP1A1-, 1A2-, and 1B1- mediated reduction is still lacking. Therefore, this feature is the aim of the present study.
Molecular modeling capable of evaluating interactions of AAI with the active site of human CYP1A1, 1A2 and 1B1 under the reductive conditions was used. In silico docking, employing soft-soft (flexible) docking procedure was used to study the interactions of AAI with the active sites of these human enzymes.
The predicted binding free energies and distances between an AAI ligand and a heme cofactor are similar for all CYPs evaluated. AAI also binds to the active sites of CYP1A1, 1A2 and 1B1 in similar orientations. The carboxylic group of AAI is in the binding position situated directly above heme iron. This ligand orientation is in CYP1A1/1A2 further stabilized by two hydrogen bonds; one between an oxygen atom of the AAI nitro-group and the hydroxyl group of Ser122/Thr124; and the second bond between an oxygen atom of dioxolane ring of AAI and the hydroxyl group of Thr497/Thr498. For the CYP1B1:AAI complex, however, any hydrogen bonding of the nitro-group of AAI is prevented as Ser122/Thr124 residues are in CYP1B1 protein replaced by hydrophobic residue Ala133.
The experimental observations indicate that CYP1B1 is more than 10× less efficient in reductive activation of AAI than CYP1A2. The docking simulation however predicts the binding pose and binding energy of AAI in the CYP1B1 pocket to be analogous to that found in CYP1A1/2. We believe that the hydroxyl group of S122/T124 residue, with its polar hydrogen placed close to the nitro group of the substrate (AAI), is mechanistically important, for example it could provide a proton required for the stepwise reduction process. The absence of a suitable proton donor in the AAI-CYP1B1 binary complex could be the key difference, as the nitro group is in this complex surrounded only by the hydrophobic residues with potential hydrogen donors not closer than 5 Å.
已证明源自马兜铃属植物的草药马兜铃酸（AA）是马兜铃酸肾病（AAN）、巴尔干地方性肾病（BEN）及其尿路上皮恶性肿瘤的病因。AAN和BEN的一个共同特征是，并非所有接触AA的个体都会患肾病和肿瘤。这些不同反应的一个原因可能是催化AA生物转化的酶活性存在个体差异。因此，鉴定主要参与AA主要毒性成分AAI代谢的酶，并详细了解其催化特异性至关重要。已发现人细胞色素P450（CYP）1A1和1A2酶负责AAI的还原激活以形成AAI-DNA加合物，而其结构相关类似物CYP1B1几乎没有这种活性。然而，仍缺乏对CYP1A1、1A2和1B1介导的还原机制细节差异的了解。因此，本研究旨在探讨这一特征。
采用能够评估还原条件下AAI与人CYP1A1、1A2和1B1活性位点相互作用的分子建模方法。采用软-软（柔性）对接程序进行计算机模拟对接，以研究AAI与这些人源酶活性位点的相互作用。
在所有评估的CYP中，预测的AAI配体与血红素辅因子之间的结合自由能和距离相似。AAI也以相似的方向结合到CYP1A1、1A2和1B1的活性位点。AAI的羧基处于直接位于血红素铁上方的结合位置。在CYP1A1/1A2中，这种配体方向通过两个氢键进一步稳定；一个氢键存在于AAI硝基的氧原子与Ser122/Thr124的羟基之间；另一个氢键存在于AAI二氧戊环的氧原子与Thr497/Thr498的羟基之间。然而，对于CYP1B1:AAI复合物，由于CYP1B1蛋白中的Ser122/Thr124残基被疏水残基Ala133取代，AAI硝基的任何氢键形成都被阻止。
实验观察表明，CYP1B1在AAI还原激活方面的效率比CYP1A2低10倍以上。然而，对接模拟预测AAI在CYP1B1口袋中的结合姿势和结合能与在CYP1A1/2中发现的类似。我们认为，S122/T124残基的羟基，其极性氢靠近底物（AAI）的硝基，在机制上很重要，例如它可以为逐步还原过程提供所需的质子。AAI-CYP1B1二元复合物中缺乏合适的质子供体可能是关键差异，因为在该复合物中硝基仅被疏水残基包围，潜在的氢供体距离不小于5埃。
