Enhancing CO₂ fixation catalyzed by salicylic acid decarboxylase with remodeled tunnels

Excessive CO₂ emissions have led to increasingly severe climate and environmental challenges. Within the framework of carbon neutrality, biological carbon fixation is a promising approach to improve the ecological environment and optimize energy systems. Salicylic acid decarboxylase (SAD) catalyzes the conversion of CO₂ and phenolic compounds into salicylic acid derivatives. However, the efficiency of CO₂ fixation in this process is low and is limited by the difficulty of product release during carboxylation. In this study, the engineered SAD, with the tunnel microenvironment remodeled to increase the turnover number (k_cat), exhibited heightened CO₂ fixation efficiency. The mutant K23A/Y64T introduces a new tunnel architecture that indirectly enlarges the bottleneck radius of the native tunnel. These changes in the tunnel microenvironment facilitated product release, which was supported by evidence from both the catalytic activity and random acceleration molecular dynamics (RAMD). Kinetic analysis revealed that K23A/Y64T exhibited a 26-fold increase in catalytic efficiency (k_cat/K_m) with a K_m consistent with that of the wild type, and the CO₂ conversion efficiency increased 14-fold. Dynamic cross-correlation matrices (DCCM) illustrated that α1 with K23A and loop5 with Y64T performed anticorrelated movements, enabling a change in the tunnel microenvironment between the two regions. Unlike conventional approaches targeting substrate-enzyme interactions, this study demonstrates that protein tunnel remodeling can intrinsically enhance carboxylation activity and holds potential for broad substrate applicability.