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所有21个细胞色素P450(细胞色素P450，Cyps)。第一阶段的目的是(1)从新的细胞色素P450家族中鉴定新的反应类型；(2)研究新的细胞色素P450的功能；(3)研究具有潜在生理功能的细胞色素P酶；(4)深入了解粘细菌细胞色素P450的反应机制的结构基础。获得的主要结果如下：1)对已知的第一个类固醇1α-羟基酶(CYP260A1)进行了鉴定，并对其三维结构进行了测定。2)CE56环糊精是一种高效、高选择性的萜类羟基酶，是一种有趣的萜类修饰新工具。3)建立了环氧化反应的高效氧化还原体系，并在环氧化反应中发现了新的反应。4)有效地羟化药物，得到与人类相同的产物。1)虽然首次发现粘细菌的胞外多糖参与类去甲异戊二烯和倍半萜的转化，但对其底物空间的研究还不够深入，无法理解催化反应的多样性。了解参与这些反应的细胞色素P450(如：CYP260A1、CYP264B1)的构效关系有助于为不同类型的萜类羟化反应建立工具箱。这一点特别令人感兴趣，因为在已知的~14,000个倍半萜类化合物中，大部分是被氧化的，最常见的氧化是由Cyps催化的。2)与此相关的是，目前尚未鉴定出的两个在纤维链霉菌SOCE56基因组中发现的倍半萜环化酶的特性以及所产生的萜类化合物的进一步修饰还有待完成。3)根据已获得的X-射线数据，需要对迄今鉴定的第一个类固醇1α-羟基酶此外，基于初步的X射线数据，应该解析CYP109D1的结构，以便与通过突变获得的新功能数据的关联成为可能。4)将进行一种新的雄烯二酮衍生物的化学-酶法合成，该衍生物可能有助于抑制芳香酶-这种方法用于乳腺癌的治疗-并开辟了一种创新的方法，将细菌环氧合酶对类固醇的羟基化与化学合成相结合。对于所有任务，在过去几年中已经取得了基本结果，并为项目的成功完成奠定了基础，届时将对结果进行额外的实验和数据处理

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