Enzymatic(de)carboxylation Mechanism Of Reversible Conversion Of 2-furoic Acid To 2,5-furandicarboxylic Acid: A Theoretical Study

Dwindling fossil fuels currently threaten humanity’s survival under the rapid development of modern industry,along with sharp increasing carbon emissions.Therefore,carboxylation has received much attention as a reaction that fixes CO₂ and produces chemicals at the same time.However,only a few chemical-based methods for CO₂ fixation have been applied in industrial production because the activation of substrates always requires high energy input.Compared with chemical processes,enzymatic conversion of CO₂ has advantages of green,mild,and excellent efficiency/selectivity,which shows substantial application prospects for solving energy shortage and environmental issues.However,all enzymatic carboxylation reactions mentioned above are difficult to use in large-scale production due to their low substrate specificity and the requirement of ATP activation.Therefore,the prenylated flavin（pr FMN）dependent Ubi D family of reversible（de）carboxylases have great potential to catalyze（de）carboxylation efficiently.HmfF from P.thermopropionicum（PtHmfF）,which belongs to the Ubi D family,is proposed to catalyze reversible（de）carboxylation reactions between 2,5-furandicarboxylic acid（FDCA）and 2-furoic acid in vivo.FDCA is similar to that of petroleum-based monomer terephthalic acid（PTA）and can synthesize degradable high-performance polyesters.Therefore,PtHmfF has considerable research value both at the level of CO₂ fixation and the production of high-value-added products.However,the thermodynamics of the reactions greatly favor the decarboxylation of FDCA but limit the carboxylase activity under physiological conditions.A lack of understanding of mechanistic differences between the decarboxylation and carboxylation reactions also hinders the modification of PtHmfF in the direction of enhanced carboxylation capacity.For these reasons,we discussed in depth the complete binding process of FDCA/2-furoic acid substrates to the catalytic center and the mechanism of decarboxylation/carboxylation reactions catalyzed by PtHmfF.Molecular dynamics simulations were used to investigate the key residues that affect the binding of the substrate during the PtHmfF-catalyzed decarboxylation and carboxylation reactions.We have found that the FDCA binding into PtHmfF dimer is critically affected by a limited hinge motion and a steric hindrance of residue L166 at the entrance of the catalytic center.In contrast,the binding of furoic acid or carbon dioxide is not a rate-limiting factor in the carboxylation reaction due to its easy access to the catalytic center.Furthermore,we have investigated the reversible（de）carboxylation mechanism with the substrate of FDCA and furoic acid,respectively.When FDCA is catalyzed in the decarboxylation reaction,a wheland-type intermediate formed by a nucleophilic attack participates in the complete reaction steps.For the carboxylation reaction,PtHmfF employs a 1,3-dipolar cycloaddition mechanism to form a five-membered pr FMN-furoic acid intermediate in the first step.While the subsequent proton transfer from the Cαatom of the intermediate to E259,accomplished after the ring cleavage occurred,is rate-limiting with an energy barrier of 23.5 kcal/mol.In addition,the proton transfer steps in the decarboxylation and carboxylation reactions exhibit a significant difference in energy barriers.Through quantum chemical calculations,we found that the cause of this phenomenon is the larger p Ka value of 2-furoic acid in the intermediate compared to residue E259,while the proton is more difficult to ionize.In summary,this study demonstrated that the decarboxylation and carboxylation reactions catalyzed by PtHmfF are not strictly reversible and confirmed that the determining factor of carboxylation efficiency is the high energy barrier of proton transfer in the catalytic process rather than the substrate binding process.Further,the above findings provided the theoretical foundation for designing PtHmfF with high carboxylation activity based on transition state theory.