Characterization of the potential of selected monooxygenases of Sorangium cellulosum So ce56 for the biosynthesis of complex compounds and analysis of the reaction mechanisms

我们已经鉴定并克隆了纤维堆囊菌So ce 56中发现的所有21种细胞色素P450（CYP）。第一个项目阶段的目的是（i）从新的细菌家族中鉴定CYP的新反应类型，（ii）研究新CYP的功能，（iii）研究具有潜在有趣生理功能的CYP，以及（iv）深入了解粘细菌CYP反应机制的结构基础。主要结果如下：1）对第一个已知的甾体1 α-羟化酶（CYP 260 A1）进行了表征，并对其三维结构进行了测定。2)因此，ce 56 CYP是一种高效的、高选择性的萜烯羟化酶，显示了它们作为萜烯修饰的有趣的新工具。3)建立了一个高效的氧化还原体系，并发现了新的反应，埃坡霉素的CYP酶从So ce 56 4）So ce 56 CYP有效羟基化药物，导致产品与人的相同。以下重要的开放性问题仍然存在：1）虽然首次发现粘细菌的CYP参与了类降异戊二烯和倍半萜的转化，但尚未对其底物空间进行充分研究以了解催化反应的多样性。了解参与这些反应的CYP（例如CYP 260 A1，CYP 264 B1）的结构-活性关系可以帮助创建各种类型的萜烯羟基化的工具箱。这是特别令人感兴趣的，因为在约14，000种已知的倍半萜类化合物中，大部分被氧化，并且最常见的氧化是由CYP催化的。2)与此相关，我们对在S. cellulosum So ce 56的合成以及CYP对所产生的萜烯的进一步修饰必须完成。3)基于已经获得的X射线数据，CYP 260 A1的3D结构，这是迄今为止表征的第一种类固醇1 α-羟化酶，以及它的近亲CYP 260 B1，它以较低的选择性执行11-脱氧皮质醇和其他类固醇的羟基化，应该得到解决，并将更深入地了解细菌CYP的类固醇羟基化的结构基础。此外，基于初步的X射线数据的结构的CYP 109 D1应解决，使与新的功能数据获得的突变体的CYP 109 D1的相关性成为可能。4)一种新的雄烯二酮衍生物的化学-酶促合成，其可用于抑制芳香酶-并且这种方法用于治疗乳腺癌-将被执行，并且开辟了一种创新的方式来结合细菌CYP和化学合成的类固醇的联合收割机羟基化。对于所有任务，在过去几年中已经取得了基本成果，并为项目的成功完成奠定了基础，届时将进行额外的实验和结果的数据处理

We have identified and cloned all 21 cytochromes P450 (CYPs) found in Sorangium cellulosum So ce56. The aim of the first project phase was (i) to identify new reaction types of CYPs from novel CYP families, (ii) to study the function of novel CYPs, (iii) to investigate CYPs with potentially interesting physiological functions and (iv) to get insight into the structural basis of the reaction mechanism of myxobacterial CYPs. The following main results were obtained: 1) The first steroid 1alpha-hydroxylase (CYP260A1) known to date was characterized; determination of its 3D structure is underway. 2) So ce56 CYPs are efficient and highly selective terpene hydroxylases displaying them as interesting new tools for terpene decoration. 3) A highly efficient redox system for epothilone epoxidation was established and novel reactions on epothilones were found using CYPs from So ce56 4) So ce56 CYPs efficiently hydroxylate drugs leading to products identical to those found in human. The following important open questions remained: 1) Although it was found for the first time that the CYPs from myxobacteria are involved in the conversion of norisoprenoids and sesquiterpenes, their substrate space has not been investigated sufficiently to understand the variety of catalyzed reactions. Understanding the structure-activity relationships of CYPs (e.g. CYP260A1, CYP264B1) involved in these reactions could help to create a toolbox for various types of terpene hydroxylations. This is of special interest, since among the ~14,000 known sesquiterpenoids, a major part is oxidized and the most common oxidations are catalysed by CYPs. 2) In connection with this, the characterization of the two so far uncharacterized sesquiterpene cyclases found in the genome of S. cellulosum So ce56 and the further decoration of the produced terpenes by CYPs has to be finished. 3) Based on already obtained X-ray data, the 3D structures of CYP260A1, which is the first steroid 1alpha-hydroxylase characterized so far, and of its close relative, CYP260B1, which performs a hydroxylation of 11-deoxycortisol and other steroids with lower selectivity, should be resolved and will give deeper insight into the structural basis of steroid hydroxylation by bacterial CYPs. In addition, based on preliminary X-ray data the structure of CYP109D1 should be resolved so that correlations with novel functional data obtained with mutants of CYP109D1 become possible. 4) A chemo-enzymatic synthesis of a novel androstenedione derivative, which could be useful for the inhibition of aromatase -and this way for the treatment of breast cancer- will be performed and opens up an innovative way to combine hydroxylation of steroids by bacterial CYPs and chemical synthesis. For all tasks, fundamental results have already been obtained during the past years and form the basis for a successful finish of the project when additional experiments and data processing of the results will be performed