title: Engineering thermostable (R)-selective amine transaminase from Aspergillus terreus through in silico design employing B-factor and folding free energy calculations.
abstract: Amine transaminases have recently gained a lot of attention for the synthesis of chiral amines. Using (R)-selective amine transaminase from Aspergillus terreus (AT-ATA) as a transaminase model, in silico design was applied employing B-factor and folding free energy (ΔΔGfold) calculations. Mutation sites were selected by targeting flexible regions with the greatest B-factors, and were substituted with amino acids that were determined by folding free energy calculations (ΔΔGfold< 0) to be more rigid than the original ones. By site-directed mutagenesis, we obtained four stabilized mutants (T130M, T130F, E133F and D134L) with improved stability from 19 candidates. Compared to the wild type, the best single mutant (T130M) showed an increase in thermal stability with a nearly 2.2-fold improvement of half-life (t1/2) at 40 °C and a 3.5 °C higher T1/210 min. The optimum catalytic temperature of T130F was increased by 10 °C. In addition, the T130M/E133F double mutant displayed the largest shift in thermostability with 3.3-fold improvement of t1/2at 40 °C and a 5.0 °C higher T1/210 min. Modeling analysis showed that new hydrophobic interactions and hydrogen bonds might contribute to the observed thermostability improvement.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China. Electronic address: hjunlzr@163.com.
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.affiliation: School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China.
authors.forename: Dong-Fang
authors.initials: DF
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China. Electronic address: yfeng2009@sjtu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
mesh_terms: D000588:Amines / Q000737:chemistry / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D003198:Computer Simulation; D004795:Enzyme Stability; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013696:Temperature; D000637:Transaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000588:Amines; D011994:Recombinant Proteins; D000637:Transaminases
keywords: Amine transaminase; B-factor; Folding free energy; Hydrophobic interaction; Thermostability