Cytochrome P450 BM3 (BM3) is an important oxidoreductase that is widely used in drug synthesis, chemical synthesis, and other industries. However, as BM3 unquestionably increases costs by consuming a natural cofactor that unstably provides electrons, an alternative biomimetic cofactor with simpler structures represented by nicotinamide mononucleotide (NMNH) has been utilized. Currently, few reports exist on artificially modified BM3 enzymes using NMNH, especially regarding theoretical simulation and calculation. With the cognition of the mechanism in mind, we propose a strategy that optimizes and refines catalytic conformation. Based on constrained molecular dynamics simulation, the distance between N-5 of FAD flavin and C-4 of NMNH is used as a cue for the determination of improved conformation, and the potential positive mutants are subsequently screened virtually in accordance with binding free energy requirements. As a result, the K cat/K M values of the favorable mutant S848R increased to 205.38% compared to the wild-type BM3 with NMNH. These data indicate that our strategy can be applied for the specific utilization of biomimetic cofactors by oxidoreductases represented by BM3.
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