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Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity
Author(s) -
Wang Xinye,
Jing Xiaoran,
Deng Yi,
Nie Yao,
Xu Fei,
Xu Yan,
Zhao YiLei,
Hunt John F.,
Montelione Gaetano T.,
Szyperski Thomas
Publication year - 2020
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.13652
Subject(s) - saturated mutagenesis , pullulanase , protein engineering , mutagenesis , directed evolution , site directed mutagenesis , biology , genetics , computational biology , biochemistry , enzyme , mutation , gene , mutant
Pullulanases are well‐known debranching enzymes hydrolyzing α‐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.