Local Flexible-Region Engineering Improves the Thermostability and Catalytic Activity of 2-Deoxy-D-Ribose-5-Phosphate Aldolase.

2-Deoxy-D-ribose-5-phosphate aldolase (DERA) is a useful biocatalyst, but its application is limited by poor thermostability. Here, a flexibility-guided engineering strategy was applied to Escherichia coli DERA (EcDERA) to improve thermostability while retaining catalytic performance. Molecular dynamics simulations were used to identify flexible regions for mutation design. Among the variants tested, D26P/Y233F showed the best overall performance. Its half-life at 60 °C increased from 103 to 363 min (3.5-fold), and its specific activity increased by 60.9% (17.28 vs 10.74 U·mg-1) compared with the wild type. Structural and molecular dynamics analyses were consistent with reduced N-terminal fluctuations for D26P and changes in the local hydrophobic environment and substrate-binding interactions associated with Y233F. The double mutant also retained a catalytic efficiency comparable to that of the wild type. Targeted modification of flexible regions therefore offers a practical route to improving EcDERA thermostability while preserving catalytic performance.