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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.