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Abstract:
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.
