Computational methods for the study of carboxylases: The case of crotonyl-CoA carboxylase/reductase

The rising levels of atmospheric CO ₂ and its impact on climate change call for new methods to transform this greenhouse gas into beneficial compounds. Carboxylases have a significant role in the carbon cycle, converting gigatons of CO ₂ into biomass annually. One of the most effective and fastest carboxylases is crotonyl-CoA carboxylase/reductase (Ccr). To understand its underlying mechanism, we have developed computational methods and protocols based on all-atom molecular dynamics simulations. These methods provide the CO ₂ binding locations and free energy inside the active site, dependent on different conformations adopted by Ccr and the presence of the crotonyl-CoA substrate. Furthermore, the adaptive string method and quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations outline the CO ₂ fixation reaction via two different mechanisms. The direct mechanism involves a hydride transfer creating a reactive enolate, which then binds the electrophilic CO ₂ molecule, resulting in the carboxylated product. Alternatively, another mechanism involves the formation of a covalent adduct. Our simulations suggest that this adduct serves to store the enolate in a much more stable intermediate avoiding its reduction side reaction, explaining the enzyme's efficiency. Overall, this work presents computational methods for studying carboxylation reactions using Ccr as a model, providing general principles that can be applied to modeling other carboxylases.