Improvement of the activity and thermostability of l‑threonine aldolase from Pseudomonas putida via tailoring of the active sites lining the binding pocket  

作　　者：Lihong Li Rongzhen Zhang Wenchi Zhang Yan Xu 

机构地区：[1]Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology,Jiangnan University,Wuxi 214122,People’s Republic of China [2]Lab of Brewing Microbiology and Applied Enzymology,School of Biotechnology,Jiangnan University,Wuxi 214122,People’s Republic of China [3]Solomon H.Snyder Department of Neuroscience,Johns Hopkins University School of Medicine,Baltimore,MD 21205,USA

出　　处：《Systems Microbiology and Biomanufacturing》2023年第3期440-448,共9页系统微生物学与生物制造（英文）

基　　金：the National Key research and Development Program of China(2018YFA0900302);the National Science Foundation of China(32271487,31970045);the National Firstclass Discipline Program of Light Industry Technology and Engineering(LITE2018-12);the Program of Introducing Talents of Discipline to Universities(111-2-06);Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.

摘　　要：l-threonine aldolases catalyze the conversion of glycine and aldehydes to synthesizeβ-hydroxy-α-amino acids with unsatisfactory enzyme activity.Here,we expressed the l-threonine aldolase from Pseudomonas putida KT2440(l-PpTA)in Escherichia coli BL21(DE3)and improved the activity and thermostability by protein engineering.Five amino acid residues(Ser10,His89,Asp93,Arg177,and Arg321)located in the substrate-binding pocket were selected and for mutation.Eight mutants(D93A,D93G,D93M,D93F,D93S,D93Q,D93Y and D93H)with increased enzyme activity were identified and their k_(cat)/K_(M)values showed about 1-7-fold higher than wild-type.Among all the variants,D93H showed the highest catalytic efficiency with 2925 and 4515 s^(−1)mM^(−1)of k_(cat)/K_(M)values toward l-threonine and l-allo-threonine,respectively.In addition,circular dichroism spectrum exhibited that the melting temperature of D93H(54.2℃)was 5℃higher than wild-type(49.2℃).Molecular dynamics simulations illustrated that the D93H variant shortens the distance between the imidazole group of H93 and the hydroxyl group of substrate,which facilitated the proton extraction and promote the enzymatic reaction.This work affords a candidate for the synthesis ofβ-hydroxy-α-amino acids with improved catalytic efficiency and thermostability and provides structural insights into the l-TA family by protein engineering.

关 键 词：Threonine aldolases Pseudomonas putida Catalytic efficiency Thermostability Molecular dynamics simulations 

分 类 号：O62[理学—有机化学]