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Abstract

Anthropogenic carbon dioxide (CO2) emissions cause an imbalance in the global carbon cycle that consequently leads to global warming. Besides the indisputable role of CO2 as harmful greenhouse gas, this small molecule harbors great potential as a simple and accessible carbon source. To exploit this potential, biotechnological strategies need to be established to convert CO2 into value-added products, like fuels or antibiotics. It is therefore indispensable to identify and characterize efficient carboxylases. To date, the members of the enoyl-CoA carboxylase/reductase (Ecr) family account for the most efficient carboxylases found in nature. Their efficiency partly depends on the effective stabilization of the CO2 molecule within the active site. The conserved CO2-binding motif is characteristic for Ecrs. This work deals with the thorough study of the three-domain fusion enzyme propionyl-CoA synthase (Pcs) of Erythrobacter sp. NAP1. This complex enzyme comprises the Ecr family CO2-binding motif in its reductase domain, suggesting a potential carboxylase activity and therefore deserves detailed investigation. The first part sets a focus on the biochemical features of Pcs. Combined kinetic and structural analysis proposed that Pcs uses a highly synchronized catalytic mechanism to sequester its reactive intermediate acrylyl-CoA. X-ray crystallography revealed an enclosed reaction chamber that features all three active sites of the fusion enzyme. This allows for the catalysis of the three subsequent reactions within the chamber. Kinetic data supported the idea that conformational changes in the Pcs ligase domain regulate the opening and closing of the catalytic compartment. Additional structural elements in Pcs either mimic domains of neighboring protomers in standalone homologues that contribute essential residues for catalysis or seal the reaction chamber. The presumed carboxylation potential of the reductase domain was demonstrated albeit at a very low efficiency in Pcs wildtype. Rational design was used to implement the two principles of efficient carboxylation known from Ecrs into the Pcs reductase domain. Improved CO2-binding and shielding of the active site from water converted the reductase domain into a carboxylase domain. The engineered trifunctional, substrate-channeling carboxylase could prove advantageous in synthetic CO2-fixation pathways. In the second part of this work, light is shed on the physiological and ecological role of Pcs. While well described in the context of the autotrophic 3-hydroxypropionate bi-cycle in Chloroflexus aurantiacus, the presence of Pcs in the genome of several (potential photo-) heterotrophic microorganisms suggests an alternative function. The genome of the aerobic anoxygenic phototrophic bacterium Erythrobacter sp. NAP1 encodes homologous enzymes of a partial 3-hydroxypropionate bi-cycle able to convert acetyl-CoA and two bicarbonate molecules into succinyl-CoA. The two key enzymes, Pcs and malonyl-CoA reductase (Mcr), were shown to be upregulated when the cells were grown in the light. Hence, it was suggested that this pathway might be involved in the adjustment of photosynthesis-induced redox imbalance.