Mechanism And Rational Redesign Study Of Styrene Monooxygenase For Catalyzing Olefin Hydration Reaction

Primary alcohols are key precursors for the synthesis of many important fine chemicals and drugs,and their preparation methods have been widely studied.The olefin hydration reaction for the synthesis of primary alcohol compounds has advantages such as atom economy and redox neutrality.However,the traditional acid-catalyzed olefin hydration reaction strictly follows Markovnikov’s rule,making it impossible to obtain primary alcohols via this method.The classic borohydride-oxidation reaction can indirectly achieve the preparation of primary alcohols from olefins,but it requires stoichiometric amounts of borane and peroxide,which are expensive and pose certain hazards.Therefore,developing a mild olefin anti-Markovnikov hydration reaction has been a challenge in the field of catalysis.So far,only the styrene monooxygenase StyA has been found to have the ability to catalyze the olefin anti-Markovnikov hydration reaction,but the mechanism of its catalysis of olefin hydration is still unresolved.On the other hand,the activity of natural StyA for catalyzing olefin hydration is also low.In this thesis,the function of the residues at catalytic sites of StyA for catalyzing the anti-Markovnikov hydration reaction of olefin was carefully analyzed,and the mechanism of StyA-catalyzed anti-Markovnikov hydration reaction of olefin was clarified.In addition,the mutants with high hydration reaction reactivity and prefer hydration were obtained through rational design of the StyA substrate binding pockets and the sites related to the binding of FAD.The main research results are as follows:The key catalytic residues of StyA for catalyzing the anti-Markovnikov hydration reaction of olefins,including residue 46,100,and 309,were mutated to hydrophobic,acidic,basic,or other polar amino acids.The catalytic functions of the mutant variants were analyzed,and it was determined that the acidic amino acid at residues 100 and the basic or polar amino acids at residues 46 and 309 play important roles in the StyA-catalyzed anti-Markovnikov hydration reaction.In order to further elucidate the mechanism of the enzyme-catalyzed reaction,the products of the StyA-catalyzed hydration reaction of fluorinated styrene were analyzed.It was confirmed that a carbon anion intermediate exists in the process of StyA-catalyzed anti-Markovnikov hydration reaction of olefins.Combining with the characteristics of key catalytic amino acid residues,it was proposed that Asp100 deactivates water molecules,enabling the hydroxyl attack on the olefin and the formation of a carbon anion intermediate.Asn46 and Asn309 stabilize the carbon anion intermediate,and then the proton attacks the carbon anion intermediate,completing the Anti-Markovnikov hydration reaction between the olefin and water.In order to improve the enzymatic activity of the anti-Markovnikov hydration reaction of olefin,this paper first modified the amino acids in the substrate binding pocket of StyA,resulting in the mutant variants N46 K,N46C,H50 Q,and H51 Q,which exhibited increased activity in catalyzing the hydration reaction.The activity of these variants was 1.3,1.5,1.2,and 1.3 times that of the wild-type enzyme,respectively.In addition,mutants D100 E,Y101D,V48 K,and H50 Q were obtained with improved hydration reaction selectivity(the ratio of hydration activity to epoxidation activity > 99%).Furthermore,by rational design of StyA and FAD binding sites,mutant A49 F was obtained,which retained only the activity for anti-Markovnikov hydration reaction of olefins,and the hydration reaction selectivity of this mutant was increased to over 99%.Finally,by combining mutations at different sites,mutants N46S-A49 F,N46S-H51 Q,and A49F-H51 Q were obtained,which exhibited 1.4,2.6,and 3.8 times higher hydration reaction activity than wild-type,respectively,with the hydration selectivity of N46S-A49 F exceeding 99%.