Abstract
Developing new carbon dioxide (CO(2)) 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 CO(2) and suppressing diffusion of water to the active site. The engineered carboxylases show improved CO(2)-binding and kinetic parameters comparable to naturally existing CO(2)-fixing enzymes. Our results provide a strategy to develop novel CO(2)-fixing enzymes and shed light on the emergence of natural carboxylases during evolution.