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

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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.