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Hierarchical Design Synergizing Local Dynamics Optimization and Global Rigidification Unlocks the Catalytic Potential
Chunxiang Pu1, Tenghe Wang1, Yu Lv1
1Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Abstract:
A key challenge in enzyme engineering is coordinating local catalytic dynamics with global structural integrity. Here, we present a hierarchical strategy integrating local dynamics optimization with global scaffold rigidification, using d-erythrose-4-phosphate dehydrogenase (Epd) as a model. Guided by evolutionary and conformational network analyses, we first engineered local active-site dynamics, yielding a double mutant (G14A/A234S) with 3-fold higher kcat and improved thermostability (ΔTm = 2 °C). Global rigidification further empowered the preoptimized active site, producing a quadruple mutant (G14A/A234S/T31I/V17I) with 11.2-fold increased kcat and an additional +2 °C Tm rise. Molecular dynamics simulations revealed that distal rigidification suppresses nonproductive fluctuations and enriches catalytically competent conformations. This "inner flexibility, outer rigidity" architecture boosted vitamin B6 production by 3.9-fold. Our work establishes a mechanism-guided paradigm for synergistically enhancing both activity and stability, offering a generalizable framework for engineering industrial biocatalysts.
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