Covalent Adduct Formation as a Strategy for Efficient CO₂ Fixation in Crotonyl-CoA Carboxylases/Reductases

Abstract

Increasing levels of CO 2 in the atmosphere have led to a growing interest into the various ways nature transforms this greenhouse gas into valuable organic compounds. Crotonyl-CoA carboxylases/reductases (Ccr's) are the most efficient biocatalysts for CO 2 fixation because of their oxygen tolerance, their high catalytic rate constants, and their high fidelity. The reaction mechanism involving hydride transfer from the NADPH cofactor and addition of CO 2 to the reactive enolate, however, is not completely understood. In this study, we use computer simulations in combination with high-level ab initio calculations to trace the free energy landscape along two possible reaction paths: In the direct mechanism, hydride transfer is immediately followed by CO 2 addition, whereas in the C2 mechanism a thermodynamically stable covalent adduct between the substrate and the NADPH cofactor is formed. This C2 adduct, which has been previously characterized experimentally, serves as a stable intermediate avoiding the reduction side reaction of the reactive enolate species, and it is able to react with CO 2 with similar kinetics as the direct reaction mechanism as confirmed by measured kinetic isotope effects. Thus, our results show that nature's most efficient CO 2 -fixing enzyme uses the formation of a covalent adduct as a strategy to store the reactive enolate species. The emerging microscopic picture of the CO 2 -fixing mechanism confirms previous experimental observations and provides new insights into how nature handles highly reactive intermediates to fix this inert greenhouse gas.

Conformational Dynamics of the Most Efficient Carboxylase Contributes to Efficient CO2 Fixation

“…The preparation finishes with substrate binding to the subunit as opposed to the closed one on the same dimer­(gray). Rx: Once in the closed conformation (blue), the chemical reaction takes place following the reaction mechanism described by us previously . +CO 2 : after the reaction, this subunit (blue) opens partially.…”

“…This enolate attacks the electrophilic CO 2 generating the carboxylated product (2 S )-ethylmalonyl-CoA. Alternatively, it also forms a C2 adduct to avoid unfavorable side reactions with protons and store the reactive enolate as we have recently shown …”

“…Recently, we studied the reaction mechanism used by Ccr to add CO 2 to its crotonyl-CoA substrate combining computational chemistry with biochemical characterization of the reaction kinetics and X-ray structures of the apo and holoenzyme in the catalytic cycle. ,, The catalytic competent Ccr oligomer is a tetramer in a dimer of dimers configuration. Each dimer presents an open and closed subunit, as shown in Figure A.…”

“…A conserved water molecule acts as a bridge between Glu171 and His365 keeping both residues at a close distance (6 Å) in comparison to the open active site (9 Å) (see Figure B,C). The proximity of Glu171 alters the p K a of His365 resulting in different protonation states at an experimental condition of pH 8 . Protonation changes of His365 are also induced by different conformations of the tetrameric enzyme …”