Engineering the flexibility of the β-sheet containing Y58 in L-aspartate-α-decarboxylase to relieve mechanism-based...

Aspartate-α-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, while was restricted for applications by its mechanism-based inactivation. Here the flexibility of the β-sheet containing Y58, a critical residue contributing to the mechanism-based inactivation, was rationally engineered for reducing its interaction with another β-sheet which contained catalytic residue S25. For the variants, enzyme activities of variant P61A, P61K, and R64P exhibited over 75% higher than that of wild type. Notably, comparing with wild type, variant P61K showed 86.3% increase in residual enzyme activity after continuous reaction at 37℃ for 2 h. After 12 h whole-cell biocatalysis, the β-alanine titer reached 121.1 g/L for variant P61K, 145% higher than that using wild type. Molecular dynamics simulations indicated that variant P61K exhibited increased rigidity in the β-sheet containing Y58. Overall, this study offered a novel strategy to relieve the mechanism inactivation of ADC by reducing interactions between enzymatic secondary structures, which might minimize erroneous protonation of the S25 acetone group by Y58.

The online version contains supplementary material available at 10.1007/s13205-026-04924-7.