Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases

Abstract

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO₂) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO₂ molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO₂ within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO₂-fixing enzyme. Together, these 4 residues anchor and position the CO₂ molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO₂ fixation. Altogether, our study reveals unprecedented molecular details of selective CO₂ binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO₂-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO₂ in biology and chemistry.

Keywords: CO2 fixation; RuBisCO; carbon dioxide; carboxylases; enzyme mechanisms.