Skarydová Lucie, Skarka Adam, Novotná Romana, Zivná Lucie, Martin Hans-Jörg, Wsól Vladimír, Maser Edmund
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic.
Toxicology. 2009 Oct 1;264(1-2):52-60. doi: 10.1016/j.tox.2009.07.013. Epub 2009 Jul 25.
Carbonyl reducing enzymes play important roles in the biotransformation and detoxification of endo- and xenobiotics. They are grouped into two protein superfamilies, the short-chain dehydrogenases (SDR) and aldo-keto reductases (AKR), and usually are present in the cytoplasm of a cell. So far, only one membraneous carbonyl reductase has been described, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is located in the endoplasmic reticulum and which significantly contributes to the metabolism of a variety of carbonyl containing drugs and toxicants. Oracin is a new and prospective anticancer drug bearing a prochiral carbonyl moiety. The main metabolic pathway of oracin is carbonyl reduction to 11-dihydrooracin (DHO) which, however, eliminates the therapeutic potential of the drug, because the two DHO enantiomers formed have significantly less anti-tumor activities. Therefore, the oracin inactivating enzymes should urgently be identified to search for specific inhibitors and to enhance the chemotherapeutic efficacy. Interestingly, the calculation of enzyme specific activities and stereospecificities of (+)-DHO and (-)-DHO formation strongly suggested the existence of a second, hitherto unknown microsomal oracin carbonyl reductase in human liver. Therefore, the aim of the present study was to provide proof for the existence of this new enzyme and to develop a purification method for further characterization. First, we succeeded in establishing a gentle solubilization technique which provided a favourable detergent surrounding during the further purification procedure by stabilizing the native form of this fragile protein. Second, we could partially purify this new microsomal carbonyl reductase by a two step separation on Q-sepharose followed by Phenyl-sepharose. The enzyme turned out to be NADPH specific, displaying kinetic values for oracin carbonyl reduction of K(m)=42 microM and V(max)=813 nmol/(30 min x mg protein). Compared to the microsomal fraction, the enzyme specific activity towards oracin could be enhanced 73-fold, while the stereospecificity of (+)-DHO formation shifted from 40% to 86%. Considering these data for 11beta-HSD1, as described in previous reports, it is clear that the microsomal carbonyl reductase investigated in the present study is new and has a great potential to significantly impair the chemotherapy with the new anticancer drug oracin.
羰基还原酶在生物体内内源性和外源性物质的生物转化及解毒过程中发挥着重要作用。它们被分为两个蛋白质超家族，即短链脱氢酶（SDR）和醛酮还原酶（AKR），通常存在于细胞的细胞质中。到目前为止，仅有一种膜性羰基还原酶被描述，即11β-羟基类固醇脱氢酶1型（11β-HSD1），它位于内质网中，对多种含羰基药物和毒物的代谢有显著贡献。奥拉辛是一种带有前手性羰基部分的新型潜在抗癌药物。奥拉辛的主要代谢途径是羰基还原为11-二氢奥拉辛（DHO），然而，这消除了该药物的治疗潜力，因为形成的两种DHO对映体的抗肿瘤活性显著降低。因此，迫切需要鉴定出使奥拉辛失活的酶，以寻找特异性抑制剂并提高化疗效果。有趣的是，对（+）-DHO和（-）-DHO形成的酶比活性和立体特异性的计算强烈表明，人肝脏中存在第二种迄今未知的微粒体奥拉辛羰基还原酶。因此，本研究的目的是为这种新酶的存在提供证据，并开发一种纯化方法以进行进一步表征。首先，我们成功建立了一种温和的增溶技术，通过稳定这种脆弱蛋白质的天然形式，在进一步的纯化过程中提供了有利的去污剂环境。其次，我们通过在Q-琼脂糖上进行两步分离，随后在苯基琼脂糖上进行分离，部分纯化了这种新的微粒体羰基还原酶。结果表明该酶对NADPH具有特异性，对奥拉辛羰基还原的动力学值为K（m）=42μM，V（max）=813 nmol/（30 min×mg蛋白质）。与微粒体部分相比，该酶对奥拉辛的比活性可提高73倍，而（+）-DHO形成的立体特异性从40%变为86%。考虑到先前报道中关于11β-HSD1的这些数据，很明显本研究中所研究的微粒体羰基还原酶是新的，并且极有可能显著影响新型抗癌药物奥拉辛的化疗效果。
