title: Structural analysis of β-glucosidase mutants derived from a hyperthermophilic tetrameric structure.
abstract: β-Glucosidase from Pyrococcus furiosus (BGLPf) is a hyperthermophilic tetrameric enzyme which can degrade cellooligosaccharides to glucose under hyperthermophilic conditions and thus holds promise for the saccharification of lignocellulosic biomass at high temperature. Prior to the production of large amounts of this enzyme, detailed information regarding the oligomeric structure of the enzyme is required. Several crystals of BGLPf have been prepared over the past ten years, but its crystal structure had not been solved until recently. In 2011, the first crystal structure of BGLPf was solved and a model was constructed at somewhat low resolution (2.35 Å). In order to obtain more detailed structural data on BGLPf, the relationship between its tetrameric structure and the quality of the crystal was re-examined. A dimeric form of BGLPf was constructed and its crystal structure was solved at a resolution of 1.70 Å using protein-engineering methods. Furthermore, using the high-resolution crystal structural data for the dimeric form, a monomeric form of BGLPf was constructed which retained the intrinsic activity of the tetrameric form. The thermostability of BGLPf is affected by its oligomeric structure. Here, the biophysical and biochemical properties of engineered dimeric and monomeric BGLPfs are reported, which are promising prototype models to apply to the saccharification reaction. Furthermore, details regarding the oligomeric structures of BGLPf and the reasons why the mutations yielded improved crystal structures are discussed.
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakabayashi
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Misumi
authors.initials: M
authors.lastname: Kataoka
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Mishima
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Maeno
authors.affiliation: Biomass Refinery Research Center, National Institute of Advanced Industrial Science, 3-11-32, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D002152:Calorimetry, Differential Scanning; D003460:Crystallization; D018360:Crystallography, X-Ray; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics*; D013816:Thermodynamics; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D021122:Protein Subunits; D001617:beta-Glucosidase
keywords: biomass; crystal engineering; intermolecular interactions; protein engineering; thermostable enzymes