Novel multi-site enzymes in the transformation of aliphatic and aromatic hydrocarbons

为了在缺氧条件下维持生命，我们将对参与脂肪族和芳香族碳氢化合物转化的复杂多位酶的结构和功能进行分析：(1)我们将揭示单体钼蝶呤/铁-S酶乙炔水合酶(AH)将乙炔转化为乙醛的作用机理；(2)我们将开发一个基于结构的双功能THDP/FAD酶-环己烷-1，2-二酮水解酶(CDH)，它在第二步裂解环状α-二酮形成己二酸酯。研究了钼杂二聚体/铁-S酶邻苯三酚-间苯三酚转移羟基酶(TH)的反应机理。对这些复杂的多位点酶进行了纯化和鉴定。X射线结晶学和光谱技术帮助绘制了催化中心的详细图景。天然蛋白质的三维结构将为在分子水平上通过结构、光谱和动力学技术研究底物的活化/结合和转化提供良好的基础。蛋白质变体的设计将扩大底物光谱，提高催化效率，并改善催化剂的稳健性。此外，将在SPP内调查新的微生物来源，以提供相关的酶活性。

We aim at the structural and functional analysis of complex multi-site enzymes involved in the transformation of aliphatic and aromatic hydrocarbons, to maintain life under anoxic conditions: (1) we will unravel the mechanism of action of the monomeric molybdopterin/Fe-S enzyme Acetylene Hydratase (AH) which converts acetylene to acetaldehyde, (2) we will develop a structure-based reaction mechanism of the bifunctional ThDP/FAD enzyme Cyclohexane-1,2-dione Hydrolase (CDH) which cleaves a cyclic α-diketone to form adipate in a second step, and (3) we will investigate the reaction mechanism of the heterodimeric molybdopterin/Fe-S enzyme Pyrogallol-Phloroglucinol Transhydroxylase (TH). These complex multi-site enzymes have been purified and characterized. X-ray crystallography and spectroscopic techniques helped to draw a detailed picture of the catalytic centers. The 3D-structures of the native proteins will provide an excellent basis to study substrate activation/binding and transformation at the molecular level, through structural, spectroscopic, and kinetic techniques. Protein variants will be designed to enlarge the substrate spectrum, to improve the catalytic efficiency, and to improve the robustness of the catalyst. In addition, new microbial sources will be investigated within the SPP which will provide related enzymatic activities.