title: The structural analysis and the role of calcium binding site for thermal stability in mannanase.
abstract: Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K(a) of 3.02 × 10(4) M(-1) and 1.52 × 10(4) M(-1), respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.
authors.affiliation: Okayama Prefectural Technology Center for Agriculture, Forestry and Fisheries, Research Institute for Biological Sciences, 7549-1 Kibichuo-cho, Kaga-gun, Okayama 716-1241, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Kayoko
authors.initials: K
authors.lastname: Kawakami
authors.forename: Takafumi
authors.initials: T
authors.lastname: Mukaihara
authors.forename: Masayo
authors.initials: M
authors.lastname: Kimura
authors.forename: Tadashi
authors.initials: T
authors.lastname: Hatanaka
mesh_terms: D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D020134:Catalytic Domain; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D044902:beta-Mannosidase; D002118:Calcium