title: The N-terminal β-sheet of the hyperthermophilic endoglucanase from Pyrococcus horikoshii is critical for thermostability.
abstract: The β-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeon Pyrococcus horikoshii (EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with β-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (β/α)(8)-barrel fold with an N-terminal antiparallel β-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal β-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.
authors.affiliation: Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Trent C
authors.initials: TC
authors.lastname: Yang
authors.forename: Steve
authors.initials: S
authors.lastname: Legault
authors.forename: Emery A
authors.initials: EA
authors.lastname: Kayiranga
authors.forename: Jyothi
authors.initials: J
authors.lastname: Kumaran
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
authors.forename: Wing L
authors.initials: WL
authors.lastname: Sung
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D017384:Sequence Deletion
chemical_list: D002480:Cellulase