Four amino acids define the CO ₂ 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 CO2molecule 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 CO2within the active site of Ecr fromKitasatospora 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 CO2molecule 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 CO2fixation. Altogether, our study reveals unprecedented molecular details of selective CO2binding 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 CO2in biology and chemistry.

Awakening a latent carbon fixation cycle inEscherichia coli

“…Furthermore, as the carboxylating activity of Gnd would be enhanced by the carbon concentrating mechanisms of algae 66 and cyanobacteria 67 , engineering these organisms to use the GED cycle could be advantageous. However, to facilitate the establishment of the GED cycle in higher plants, the affinity of Gnd towards CO2 would have to be improved, e.g., via the rational engineering of CO2 binding sites 68 , as successfully demonstrated recently in a proof-of-principle study 69 .…”

CO2‐Folded Single‐Chain Nanoparticles as Recyclable, Improved Carboxylase Mimics

“…Notably, even for N-heterocyclic double bonds (e.g. benzopyrrole and benzoxazole), the results remained delightful ( % 70 % yield, Entry 8,9). The reason for the small decline in conversion is that N-heterocycles as Lewis bases can competitively bind the borane moieties in SCNP, leading to CO 2 premature deactivation.…”

“…Natural carboxylase depends on a binding center formed by four residues to trap CO 2 . [9] In a similar fashion, the designed SCNP should contain particular functionalities capable of collectively binding and activating CO 2 . For this, we introduce complementary frustrated Lewis acidic and basic monomers, triarylborane and triarylphosphine, to the polymer side chain.…”

Atom Condensed Fukui Function for Condensed Phases and Biological Systems and Its Application to Enzymatic Fixation of Carbon Dioxide

“…To take into account the enzymatic environment, the reactive complex formed by crotonyl-CoA and NADPH was modelled inside the active cavity of Ccr using the CHARMM22 force field, based on the crystallographic structure and modelling procedure described by Stoeffel et al 45…”

“…This is accompanied with a markedly reduced average number of hydrogen bonds of the oxygen carbonyl atom of the substrate in the enzyme (0.35) compared to aqueous solution (1.62). Hydrogen bonds present in the enzyme mostly involve amino groups of the Asn85 residue which has been identified previously by us as key residue in the catalytic mechanism 45.…”