Amine transaminases(ATAs)catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines.However,the trade-off between activity and stability in enzyme engineering represents a...Amine transaminases(ATAs)catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines.However,the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs.Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs.Herein,we modified the binding pocket of co-ATA from Aspergillus terreus(AtATA)to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone.We discovered a structural switch comprising four key amino acid sites(R128,V149,L182,and L187),as well as the"best"mutant(AtATAD224K/V149A/L182 F/L187F;termed M4).Compared to the parent enzyme AtATAD224K(AtATAPa),M4 increased the catalytic efficiency(k_(cat)/K_(m)^(1-acetonaphthone),where kcatis the constant of catalytic activities and is 10.1 min^(-1),K_(m)^(1-acetonaphthoneis) Michaelis-Menten constant and is 1.7 mmol·L^(-1))and half-life(t1/2)by 59-fold to 5.9 L·min^(-1)·mmol-1and by 1.6-fold to 46.9 min,respectively.Moreover,using M4 as the biocatalyst,we converted a 20 mmol·L^(-1)aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product,(R)-(+)-1(1-naphthyl)ethylamine((R)-NEA),with 78%yield and high enantiomeric purity(R>99.5%)within 10 h.M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs.The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability.Strengthening of intramolecular interactions and expansion of the angle between the substratebinding pocket and the pyridoxal 5’-phosphate(PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity.Moreover,this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs,which exhibited 8%-22%sequence similarity with AtATA.This research has important implications for overcoming the trade-off between ATA activity and thermostability.展开更多
The interactions between several peptides with low molecular weight (guest, NH2 Arg Arg Trp Trp H 2; NH2 Arg Trp Arg Trp H 3; NH2 Trp Arg Arg Trp H 4; NH2 Arg Arg Trp Trp Trp Trp H 5; NH2 Trp Trp Arg Arg Trp Trp H 6; ...The interactions between several peptides with low molecular weight (guest, NH2 Arg Arg Trp Trp H 2; NH2 Arg Trp Arg Trp H 3; NH2 Trp Arg Arg Trp H 4; NH2 Arg Arg Trp Trp Trp Trp H 5; NH2 Trp Trp Arg Arg Trp Trp H 6; NH2 Arg Arg Trp Trp Trp Trp Trp Trp H 7; NH2 Arg Arg Trp Trp Trp Trp Trp Trp Trp Trp H 8) and β cyclodextrin dimer (host, 1) bridged with the derivative of (1R, 3R) 1 aminocyclobutane cis 1,3 dicar boxylic acid were investigated by using fluorescence polarization method in buffer aqueous solution (pH 7.4) at 298K. The binding constants of the cyclodextrin dimer 1 to the guests 2 8 were determined. It was shown that there was a cooperative action of the two cavities of a cyclodextrin dimer in the binding of large substrates, and that the structure and properties of amino acid in the peptides played very important roles in the synergic complexation between host and guest.展开更多
基金National Natural Science Foundation of China(32071268 and 31971372)the Ningbo"Scientific and Technological Innovation 2025"Key Project(2020Z080)for financial support。
文摘Amine transaminases(ATAs)catalyze the asymmetric amination of prochiral ketones or aldehydes to their corresponding chiral amines.However,the trade-off between activity and stability in enzyme engineering represents a major obstacle to the practical application of ATAs.Overcoming this trade-off is important for developing robustly engineered enzymes and a universal approach for ATAs.Herein,we modified the binding pocket of co-ATA from Aspergillus terreus(AtATA)to identify the key amino acid residues controlling the activity and stability of AtATA toward 1-acetonaphthone.We discovered a structural switch comprising four key amino acid sites(R128,V149,L182,and L187),as well as the"best"mutant(AtATAD224K/V149A/L182 F/L187F;termed M4).Compared to the parent enzyme AtATAD224K(AtATAPa),M4 increased the catalytic efficiency(k_(cat)/K_(m)^(1-acetonaphthone),where kcatis the constant of catalytic activities and is 10.1 min^(-1),K_(m)^(1-acetonaphthoneis) Michaelis-Menten constant and is 1.7 mmol·L^(-1))and half-life(t1/2)by 59-fold to 5.9 L·min^(-1)·mmol-1and by 1.6-fold to 46.9 min,respectively.Moreover,using M4 as the biocatalyst,we converted a 20 mmol·L^(-1)aliquot of 1-acetonaphthone in a 50 mL scaled-up system to the desired product,(R)-(+)-1(1-naphthyl)ethylamine((R)-NEA),with 78%yield and high enantiomeric purity(R>99.5%)within 10 h.M4 also displayed significantly enhanced activity toward various 1-acetonaphthone analogs.The related structural properties derived by analyzing structure and sequence information of robust ATAs illustrated their enhanced activity and thermostability.Strengthening of intramolecular interactions and expansion of the angle between the substratebinding pocket and the pyridoxal 5’-phosphate(PLP)-binding pocket contributed to synchronous enhancement of ATA thermostability and activity.Moreover,this pocket engineering strategy successfully transferred enhanced activity and thermostability to three other ATAs,which exhibited 8%-22%sequence similarity with AtATA.This research has important implications for overcoming the trade-off between ATA activity and thermostability.
文摘The interactions between several peptides with low molecular weight (guest, NH2 Arg Arg Trp Trp H 2; NH2 Arg Trp Arg Trp H 3; NH2 Trp Arg Arg Trp H 4; NH2 Arg Arg Trp Trp Trp Trp H 5; NH2 Trp Trp Arg Arg Trp Trp H 6; NH2 Arg Arg Trp Trp Trp Trp Trp Trp H 7; NH2 Arg Arg Trp Trp Trp Trp Trp Trp Trp Trp H 8) and β cyclodextrin dimer (host, 1) bridged with the derivative of (1R, 3R) 1 aminocyclobutane cis 1,3 dicar boxylic acid were investigated by using fluorescence polarization method in buffer aqueous solution (pH 7.4) at 298K. The binding constants of the cyclodextrin dimer 1 to the guests 2 8 were determined. It was shown that there was a cooperative action of the two cavities of a cyclodextrin dimer in the binding of large substrates, and that the structure and properties of amino acid in the peptides played very important roles in the synergic complexation between host and guest.
基金supported by the National Basic Research and Development Program of China(973 Program,2011CB710800)New Century Excellent Talents in University(NCET-11-0658)+2 种基金Natural Science Foundation of Jiangsu Province(BK2011150)the Program of Introducing Talents of Discipline to Universities(111-2-06)a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions~~
