A key residue for the substrate affinity enhancement of a thermophilic endo-polygalacturonase revealed by computational design
- 182 Downloads
Protein engineering has been a research hotspot to improve the catalytic efficiency of industrially important enzymes. In the present study, a novel computational strategy was developed to in silico screen mutants with enhanced binding interaction between enzyme and substrate as well as catalytic efficiency. Through homology modeling and molecular dynamics (MD) simulation, four key residues related to substrate binding were identified in the endo-polygalacturonase BiPG28A from Bispora sp. MEY-1. Further analyses of the conformation, hydrogen bond interactions, and binding free energy revealed that lysine at position 129 (subsite − 2) has the strongest affinity to substrate. Biochemical and calorimetry experiments confirmed the functional role of Lys129 in substrate binding through non-covalent interactions. The common role of Lys129 was also verified in another GH28 endo-polygalacturonase. Distinguished from other protein engineering strategies involving structure resolution and construction of certain enzymes, this computational strategy represents an insightful and efficient approach to develop a “designed” enzyme with significantly enhanced binding affinity and catalytic efficiency.
KeywordsEndo-polygalacturonase Binding affinity Computational design Molecular dynamics simulation Site-directed mutagenesis
This research was supported by the National Natural Science Foundation of China (31571777), the China Modern Agriculture Research System (CARS-41), and the National Key Research and Development Program of China (2016YFD0501409-02).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Berka RM, Grigoriev IV, Otillar R, Salamov A, Grimwood J, Reid I, Ishmael N, John T, Darmond C, Moisan MC, Henrissat B, Coutinho PM, Lombard V, Natvig DO, Lindquist E, Schmutz J, Lucas S, Harris P, Powlowski J, Bellemare A, Taylor D, Butler G, de Vries RP, Allijn IE, van den Brink J, Ushinsky S, Storms R, Powell AJ, Paulsen IT, Elbourne LDH, Baker SE, Magnuson J, LaBoissiere S, Clutterbuck AJ, Martinez D, Wogulis M, de Leon AL, Rey MW, Tsang A (2011) Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris. Nat Biotechnol 29:922–927CrossRefPubMedGoogle Scholar
- Bonivento D, Pontiggia D, Matteo AD, Fernandez-Recio J, Salvi G, Tsernoglou D, Cervone F, Lorenzo GD, Federici L (2008) Crystal structure of the endopolygalacturonase from the phytopathogenic fungus Colletotrichum lupini and its interaction with polygalacturonase-inhibiting proteins. Proteins 70:294–299CrossRefPubMedGoogle Scholar
- Federici L, Caprari C, Mattei B, Savino C, Di Matteo A, De Lorenzo G, Cervone F, Tsernoglou D (2001) Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein). Proc Natl Acad Sci U S A 98:13425–13430CrossRefPubMedPubMedCentralGoogle Scholar
- Matsui I, Ishikawa K, Matsui E, Miyairi S, Fukui S, Honda K (1991) Subsite structure of Saccharomycopsis α-amylase secreted from Saccharomyces cerevisiae. J Biochem 109:566–569Google Scholar
- Tounsia H, Sassia AH, Romdhanea ZB, Lajnefa M, Dupuyb JW, Lapaillerieb D, Lomenechb AM, Bonneub M, Gargouria A, Hadj-Taieb N (2016) Catalytic properties of a highly thermoactive polygalacturonase from the mesophilic fungus Penicillium occitanis and use in juice clarification. J Mol Catal B-Enzym 127:56–66CrossRefGoogle Scholar
- Tu T, Meng K, Huang H, Luo H, Bai Y, Ma R, Su X, Shi P, Yang P, Wang Y, Yao B (2014) Molecular characterization of a thermophilic endo-polygalacturonase from Thielavia arenaria XZ7 with high catalytic efficiency and application potential in the food and feed industries. J Agric Food Chem 62:12686–12694CrossRefPubMedGoogle Scholar
- van Santen Y, Benen JA, Schröter KH, Kalk KH, Armand S, Visser J, Dijkstra BW (1999) 1.68-Å crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. J Biol Chem 274:30474–30480Google Scholar
- Velázquez-Campoy A, Ohtaka H, Nezami A, Muzammil S, Freire E (2004) Isothermal titration calorimetry. Curr Protoc Cell Biol 17(8):1–17.8.24Google Scholar