The Catalytic Performance And Characterization Of Phenylalanine Aminomutase

Phenylalanine aminomutase(PAM)is a type of enzyme with MIO(4-methylene-imidazol-5-one)as a new type of endogenous cofactor,which subsequently undergoes two dehydrations to generate this potent electrophile of enzyme protein in the process of post-translation folding.MIO mediates the transposition of the amino group at the C? position of the substrate ?-phenylalanine to the C? position to produce?-phenylalanine,which completes its isomerization reaction,that is,regional selectivity,and can also catalyze the synthesis of?-phenylalanine from the mono-unsaturated double bond of trans cinnamic acid by the addition of ammonia from position C? of the intermediate product.Different sources of phenylalanine mutase stereoselectivity of differences,for example,the product catalyzed by TcPAM from taxus chinensis is R-?-phenylalanine,and the product catalyzed by PaPAM from pantoea agglomeran is S-?-phenylalanine,thus it can be seen that regioselectivity and stereoselectivity is phenylalanine amino mutase two important characteristics of enzymology.The research team completed the cloning expression of PaPAM and the characterization of enzyme properties in the early stage,and continued to clone the expression of TcPAM and explore the characterization of enzyme properties.First,the aminomutase(Tcpam)gene derived from taxus chinensis was chemically synthesized,and the recombinant plasmid pET-sumo-Tcpam was constructed and transferred into E.coli for heterologous induction and expression to prepare recombinant enzymes and enzymatic properties for preliminary study.On this basis,TcPAM and PaPAM were mutated to study their effects on the stereoselectivity and regioselectivity of PAM.The main findings are as follows:(1)The recombinant plasmid pETsumo-Tpam was successfully expressed in E.coli to obtain soluble recombinant TcPAM,and high-purity recombinant enzyme was prepared by affinity chromatography.TcPAM can catalyze the ?-phenylalanine to?-phenylalanine was analyzed by HPLC and MS.TcPAM has an enzyme activity of 4.11 U/mg under the optimal temperature of 30? and pH 9,while PaPAM has an enzyme activity of only 2.5 U/mg under the optimal conditions;Metal ions Na+,K+,Fe2+,and Ca2+ had little effect on the activity of TcPAM and PaPAM,while Cu2+and Zn2+caused significant inhibition of the activity of TcPAM and PaPAM;the surfactants CTAB,SDS,Triton X-100 and Tween 80 had little effect on the activity of recombinase TcPAM and relative enzyme activity remained above 90%,but surfactants had a greater effect on the catalytic activity of recombinase PaPAM.Comparing the enzyme activity characteristics of recombinase TcPAM and PaPAM,it was found that the two enzymes have better pH stability,recombinase PaPAM has better thermal stability,metal ions have a greater influence on the recombinase TcPAM,and surfactants on recombinase TcPAM is smaller.(2)By comparing the sequences of TcPAM and PaPAM,it was found that PaPAM 84(corresponding position is 86 phenylalanine in TcPAM)of methionine(M)and 91(corresponding position is 93 argininein in TcPAM)isoleucine(?)of phenylalanine amino deflection regioselectivity and stereoselectivity have important influence.Therefore,the two sites of TcPAM and PaPAM were mutated,and four mutants,PaPAM-M84F,PaPAM-191R,TcPAM-F86M and TcPAM-R931,were obtained,and their effects on enzyme catalytic activity were studied.PaPAM catalyzed the conversion rate of benzodiazepine to 25%,while PaPAM-M84F and PaPAM-191R could improve the catalytic efficiency of benzodiazepine to 72.1%and 27.9%,respectively.The conversion rate of TcPAM catalyzed ?-phenylalanine was 29.7%.When TcPAM-F86M and TcPAM-R931 catalyzed ?-phenylalanine,the product could not be detected.It means that the amino acids at the two positions of TcPAM and PaPAM have an influence on the regioselectivity of PAM.TcPAM,PaPAM,PaPAM-M84F,PaPAM-191R,TcPAM-F86M and TcPAM-R931 catalyze the formation of aromatic alanine with different substituents,and the production of the product was detected by HPLC to investigate the effects of different substituent groups on the recombinase regioselectivity analysis shows that the charging properties and positions of different substituents on the substrate will affect the regioselectivity of PAM.(3)Firstly,the stereostructure of product catalyzed by tcpam and papam was studied.The catalytic product was determined by HPLC and MS,and the stereostructure of the catalytic product was identified by circular dichroism.It was concluded that the stereoscopic configuration of the PaPAM catalytic product was S-?-phenylalanine,and the stereoscopic configuration of the TcPAM catalytic product was R-?-phenylalanine.The catalytic products of PaPAM-M84F,PaPAM-191R,TcPAM-F86M and TcPAM-R93I were detected by HPLC chiral analytical column,and their effects on stereoselectivity were studied.The catalytic product of PaPAM is 95%ees ?-phenylalanine,while the catalytic products of PaPAM-M84F and PaPAM-191R are 18.8%ees and 39.4%ees ?-phenylalanine,respectively.The catalytic product of TcPAM is ?-phenylalanine with 95%eeR,but the products of TcPAM-F86M and TcPAM-R931 cannot be detected.TcPAM,PaPAM,PaPAM-M84F,PaPAM-191R,TcPAM-F86M and TcPAM-R931 catalyzed substrates with different substituents.It was found that the different types and positions of the substituents on the benzene ring of the substrate also affect the stereochemistry of PAM Selective.