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

Xinye Wang, Xiaoran Jing, Yi Deng, Yao Nie, Fei Xu, Yan Xu, Yi Lei Zhao, John F. Hunt, Gaetano T. Montelione, Thomas Szyperski

Research output: Contribution to journalArticle

1 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)799-812
Number of pages14
JournalFEBS Letters
Issue number5
StatePublished - Mar 1 2020


All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology


  • activity
  • coevolving residues
  • directed evolution
  • evolutionary information
  • pullulanase
  • saturation mutagenesis

Cite this