Design Glutamate\nDehydrogenase for Nonaqueous System\nby Motifs Reassembly and Interaction Network Analysis

Glutamate dehydrogenases (GDH) serve as the key regulated\nenzyme\nthat links protein and carbohydrate\nmetabolism. Combined with motif reassembly and mutation, novel GDHs\nwere designed. Motif reassembly of thermophilic GDH and malate dehydrogenase\naims to overcome stability and activity tradeoff in nonaqueous systems.\nStructural compatibility and dynamic cooperation of the designed AaDHs\nwere studied by molecular dynamics simulation. Furthermore, multipoint\nmutations improved its catalytic activity for unnatural substrates.\nAmino acid interaction network analysis indicated that the high density\nof hydrogen-bonded salt bridges is beneficial to the stability. Finally,\nthe experimental verification determines the kinetics of AaDHs in\na nonaqueous system. The activity of Aa05 was increased by 1.78-fold\nwith ionic liquid [EMIM]BF<sub>4</sub>. This study presents the strategy\nof a combination of rigid motif assembly and mutations of active sites\nfor robust dehydrogenases with high activity in the nonaqueous system,\nwhich overcomes the activity–stability tradeoff effect.