Engineering The Biomimetic Cofactors Of NMNH For Cytochrome P450 BM3 Based On Binding Conformation Refinement

Oxidoreductases mainly catalyze the oxidation and reduction reactions of organ-isms.Including the growth,metabolism,reproduction and other life activities of organ-isms are closely related to redox reactions.Oxidoreductase,which is so important to life activities,is naturally widely used in industrial production.Cytochrome P450 BM3(BM3)is widely used for industrial drug synthesis,food processing,among other industries.P450 BM3 require natural cofactors in the cata-lyzation process.such as nicotinamide adenine dinucleotide(NAD⁺)and nicotinamide adenine dinucleotide phosphate(NADP⁺),whose reduced forms are NADH and NADPH,respectively.However,these natural cofactors are structurally complex and unstable,which undoubtedly increases the cost and obstacles of industrial production.Nowadays,many biomimetic cofactors with simpler structures,such as nicotinamide mononucleotide(NMN⁺/NMNH),have been developed and utilized.However,most wild-type enzymes either cannot utilize biomimetic cofactors,including NMNH,or ex-hibit low activity when such cofactors are utilized.In this paper,we combine theoretical calculations and experiment to artificially modify the P450 BM3 enzymes to efficiently use NMNH for catalytic reactions.With the cognition of the mechanism in mind,we propose a strategy that optimizes and re-fines 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 deter-mination of improved conformation,and the potential positive mutants are subse-quently screened virtually in accordance with binding free energy requirements.As a result,the relative enzyme activities is 2.74-fold of the wild type and the K_cat/K_M values of the favorable mutant S848R increased by 205.38%compared to the wild-type BM3with NMNH.These data indicate that our strategy can be applied for the specific utili-zation of biomimetic cofactors by oxidoreductases represented by BM3.