Rational Design Of The Product Selectivity Of P450 Monooxygenase

P450 monooxygenase is a kind of enzyme widely distributed in nature,which has been widely used in metabolism,drug synthesis and other fields due to the diversity of its catalytic reactions.Due to the limited product selectivity of natural P450 monooxygenase,it is difficult to meet the needs of industrial applications.Therefore,optimization of product selectivity of P450 monooxygenase has become an important research direction of this enzyme.The complex structure and reaction mechanism of P450 oxidase severely restricts the research on its structure and design.In view of the above problems,this paper selected the organic classical product selective reaction: The anti-Markovnikov oxidation reaction and nitro substitution reaction were studied respectively by the anti-Markovnikov rule oxidation of P450 aMOx from Labrenzia aggregate and the nitro substitution reaction of P450 TxtE H176 F BM-3R from Streptomyces Scabies.Novel P450 monooxygenases with high product selectivity was obtained,which promoted the industrial application of P450 monooxygenase.In the first chapter of this paper,the origin,definition,classification,reaction mechanism and applications of P450 monooxygenase are summarized,and the research basis and methods of this paper are briefly described.In the second chapter of this paper,the Escherichia coli heterologous expression system of P450 aMOx was constructed.Affinity chromatography was used as purification method to obtain high yield and high purity enzyme.At the same time,a method was developed to detect the enzymatic properties of P450 aMOx.The rational design of P450 aMOx was investigated on the basis of efficient heterologous expression system and enzymatic detection method.The third chapter is the rational design of P450 aMOx,and the "Butterfly" catalytic model is proposed in this chapter,which provides guidance for the product selective modification of enzyme-catalyzed anti-Markovnikov reaction.In this chapter,the possible binding conformation of P450 aMOx with styrene was obtained through homology modeling and molecular docking.Molecular dynamics simulations were carried out to confirm the key factors affecting the anti-Markovnikov product selectivity: the reaction distance and the frequency of the substrate reaction site within the reaction distance.Based on the above analysis results,MM/GBSA was used to obtain the key sites affecting the substrate to obtain the mutation A275 G,which improved the product selectivity of P450 aMOx from 81% to gt.99%.In the fourth chapter of this paper,we constructed the Escherichia coli heterologous expression system of P450 TxtE H176 F BM-3R.Affinity chromatography was used as purification method to obtain high yield and high purity enzyme.At the same time,a method was developed to detect the enzymatic properties of P450 TxtE H176 F BM-3R.The rational design of P450 TxtE H176 F BM-3R was investigated on the basis of efficient heterologous expression system and enzymology detection method.The fifth chapter is the rational design of TxtE H176F-BM 3R,and the "clock-turning" catalytic model is proposed,which can provide guidance for enzyme catalyzed nitration reaction.Based on the resolved crystal structure of TxtE H176F(PDB ID: 5D3U),this chapter uses QM to calculate the transition state mechanism of the reaction process as peroxynitrite transition state.Based on the above results,molecular dynamics simulation and virtual saturation mutation were used to design the mutations that affect the selectivity of the products of TxtE H176F-BM 3R nitro-substitution reaction,and a detection method was developed for the efficient separation and identification of nitro-L-tryptophan(NTs)at different substitution sites.Mutations I244 M,V63F,A248 F,The substrate l-tryptophan was successfully transformed from 5-nitro-L-tryptophan(5NT)to 6-nitro-L-tryptophan(6NT)with selectivity of 50%,90% and gt for 6NT,respectively.99%.