Design of robust malate dehydrogenases by assembly of motifs of halophilic and thermophilic enzyme based on interaction network

Robust oxidoreductases in non-aqueous system is promising to bridge the gap between research and industrial application. Malate dehydrogenases (MDHs) were select as model enzyme for redesign based on motif assembly study. The approach involved first selecting or building a stable framework, which subsequently served as a template for replace of functional motifs. First, for scaffold selection, dynamic conformational change of halophilic and thermophilic MDHs was studied by molecular dynamic simulations with increasing salt concentration. Second, computationally inserted the robust motifs which responded to salt concentration into the selected scaffold, and resulted novel MDHs. Third, the top-scoring designs of structure compatibility and dynamic harmony evaluation were selected for experimental verification. The experimental result indicated that the designed MDH03 had the enhanced thermostability, pH and ionic liquid tolerance. The activity of MDH03 was 1.38-fold of parent enzyme in reaction system contain [EMIM]BF4. Further study of amino acid residues interaction network explained the reason of robustness of MDH03 based on high-density salt bridges. Research also indicated that the hydrophobic contacts and pi-pi contacts of interfacial interactions of motifs play a key role for activity and stability of MDH03. This work promoted an approach to design robust dehydrogenase with high ionic liquids tolerance and promote the further application in biosensors.