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

我们的目标是对参与脂肪族和芳香族烃转化的复杂多位点酶进行结构和功能分析，以维持缺氧条件下的生命：（1）我们将阐明将乙炔转化为乙醛的单体异蝶呤/Fe-S酶乙炔水合酶（AH）的作用机制，（2）我们将开发双功能ThDP/FAD酶环己烷-1，2-二酮水解酶（CDH）的基于结构的反应机理，其在第二步中裂解环状α-二酮以形成己二酸，（3）探讨异源二聚体异源蝶呤/Fe-S酶邻苯三酚-间苯三酚转羟酶（TH）的反应机理。这些复杂的多位点酶已被纯化和表征。X射线晶体学和光谱技术有助于绘制催化中心的详细图像。天然蛋白质的三维结构将提供一个很好的基础，研究底物的活化/结合和转化在分子水平上，通过结构，光谱和动力学技术。将设计蛋白变体以扩大底物谱，提高催化效率，并提高催化剂的稳健性。此外，将在SPP内研究新的微生物来源，这将提供相关的酶活性。

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.