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Awakening the Sleeping Carboxylase Function of Enzymes: Engineering the Natural CO₂-Binding Potential of Reductases
- Bernhardsgrütter, Iria, Schell, Kristina, Peter, Dominik M., Borjian, Farshad, Saez, David Adrian, Vöhringer-Martinez, Esteban, Erb, Tobias J.
- Journal of the American Chemical Society 2019 v.141 no.25 pp. 9778-9782
- Erythrobacter, active sites, biocatalysts, bioinformatics, biotechnology, carbon dioxide, carboxylation, enzymatic reactions, enzymes, proteins, synthetic biology
- Developing new carbon dioxide (CO₂) fixing enzymes is a prerequisite to create new biocatalysts for diverse applications in chemistry, biotechnology and synthetic biology. Here we used bioinformatics to identify a “sleeping carboxylase function” in the superfamily of medium-chain dehydrogenases/reductases (MDR), i.e. enzymes that possess a low carboxylation side activity next to their original enzyme reaction. We show that propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus possess carboxylation yields of 3 ± 1 and 4.5 ± 0.9%. We use rational design to engineer these enzymes further into carboxylases by increasing interactions of the proteins with CO₂ and suppressing diffusion of water to the active site. The engineered carboxylases show improved CO₂-binding and kinetic parameters comparable to naturally existing CO₂-fixing enzymes. Our results provide a strategy to develop novel CO₂-fixing enzymes and shed light on the emergence of natural carboxylases during evolution.