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

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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