Hierarchical Design Synergizing Local Dynamics Optimization and Global Rigidification Unlocks the Catalytic Potential of d-Erythrose-4-phosphate Dehydrogenase.

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.