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Abstract:
Efficient capture and conversion of atmospheric carbon dioxide (CO2) is a prerequisite to develop a carbon-neutral, circular future economy. Carbon fixation is the process by which inorganic carbon is fixed into biomass. In Nature, enzymes called caboxlyases are able to capture atmospheric carbon dioxide under mild conditions and catalyze its incorporation into organic molecules. It is estimated that 400 Gt of CO2 are fixed annually solely by the enzyme ribulose-1,5-bisphophate-carboxylase/oxygenase (RuBisCO), the key enzyme of photosynthesis. In comparison, CO2 utilization by chemical industries accounts for only 0.1 Gt of carbon annually and utilizes pressurized CO2, which emphasizes our need to understand the molecular mechanism that allow carboxylases to selectively interact with a CO2 at atmospheric concentrations (0.04% vol) during catalysis. Enoyl-CoA carboxylases/reductases (ECRs) represent the fastest carboxylases known to date and is, in contrast to RuBisCO, completely specific for CO2. These enzymes catalyze the reductive carboxylation of enoyl-CoAs by oxidizing one equivalent of NADPH. ECRs represent a good case study for the understanding of the CO2 chemistry that carboxylases use. In this work, we try to gain a better understanding of the underlying catalytic principles that enable ECRs to achieve high catalytic rates. Initially we focus on understanding how the precise interaction between protein and CO2 takes place at the active site of ECRs. We were able to identify and assign a function to four conserved amino acid residues found at the active site of ECRs. Three residues are responsible for the precise positioning of CO2 for nucleophilic attack by the enolate intermediate. Additionally, one residue is able to shield the active site from water thereby preventing the irreversible protonation of the enolate. These two mechanistic principles are at the base of the efficient carboxylation in ECRs. The following chapter briefly describes how the enzyme is able to accept other electrophiles than CO2. We show that ECRs can utilize formaldehyde as an alternative electrophile to CO2 thereby yielding beta-hydroxy thioesters. The exquisite stereospecificity together with the vast range of small electrophiles make ECR a potential biocatalyst for the production of various α-substituted thioesters. The last two chapters of this work focus on the structural aspects of ECR catalysis. We were able to obtain four new crystal structures of an ECR from Kitasatospora setae and to propose a model for the catalytic cycle of this enzyme. We show that the communication between and within the dimers that compose the functional homotetramer is crucial for the fast catalytic rates observed in this ECR. A separate study aims at developing an in vivo directed evolution screen to improve the catalytic properties of an ECR from Burkholderia ambifaria. Our approach yields an evolved variant, with mutations distant from the active site. The observed improved catalytic supports the importance of the residues for the catalytic rate. Both studies revealed the importance of the residues at the interface of the ECR monomers by their impact on catalytic rates of this enzyme.
