Evolutionary coupling saturation mutagenesis: Coevolution-guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

FEBS Letters

article

Xinye Wang¹  Xiaoran Jing¹  Yi Deng¹  Yao Nie¹  Fei Xu¹  Yan Xu¹  Yi-Lei Zhao³  John F. Hunt⁴  Gaetano T. Montelione⁵  Thomas Szyperski⁸ 

[1] School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University;State Key Laboratory of Food Science and Technology, Jiangnan University;State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University;Department of Biological Sciences, Columbia University;Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, The State University of New Jersey;Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, The State University of New Jersey;Department of Chemistry and Chemical Biology, and Center for Biotechnology and Integrative Studies, Rensselaer Polytechnic Institute;Department of Chemistry, The State University of New York at Buffalo

关键词: activity;    coevolving residues;    directed evolution;    evolutionary information;    pullulanase;    saturation mutagenesis;   

DOI  :  10.1002/1873-3468.13652

来源: John Wiley & Sons Ltd.

【 摘 要 】

ullulanases are well-known debranching enzymes hydrolyzing a-1,6-glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the ‘evolutionary coupling saturation mutagenesis’ (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

【 授权许可】

Unknown