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<p>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&#xa0;h. After 12&#xa0;h whole-cell biocatalysis, the β-alanine titer reached 121.1&#xa0;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.</p>

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

  • 期刊论文

  • 发表日期：

    2026年6月25日

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  • Junping Zhou,
  • Feng Wang,
  • Yingli Zhao,
  • Yiran Lin,
  • Lei Chao,
  • Xueyun Feng,
  • Yihong Wang,
  • Aijuan Wu,
  • Jingwen Xie,
  • Lanlan Chen,
  • Mian Li,
  • Haiyan Zhou,
  • Zhiqiang Liu,
  • Yuguo Zheng

摘要

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.