title: The first identification of carbohydrate binding modules specific to chitosan.
abstract: Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (Tm) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)6. The Tm elevation (ΔTm) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)6 and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔTm = 13.6 °C), when (GlcN)6 was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)n; ΔGr° values were -7.8 (n = 6), -7.6 (n = 5), -7.6 (n = 4), -7.6 (n = 3), and -7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)n with the lower affinities (ΔGr° = -5.0 (n = 3) ~ -5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)6 for individual DD1 and DD2 sites (ΔGr° = -8.6 and -6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)n (n = 2~6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.
authors.affiliation: From the Department of Advanced Bioscience, Kinki University, Nara 631-8505 Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Shinya
authors.affiliation: From the Department of Advanced Bioscience, Kinki University, Nara 631-8505 Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnuma
authors.affiliation: From the Department of Advanced Bioscience, Kinki University, Nara 631-8505 Japan.
authors.forename: Reina
authors.initials: R
authors.lastname: Yamashiro
authors.affiliation: Department of Bioscience, Fukui Prefectural University, Fukui 910-1195.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Kimoto
authors.affiliation: Department of Environmental and Biotechnological Frontier Engineering, Fukui University of Technology, Fukui 910-8505, Japan, and.
authors.forename: Hideo
authors.initials: H
authors.lastname: Kusaoke
authors.affiliation: Biocenter Oulu and Department of Biochemistry, University of Oulu, Oulu, FI-90014 Finland.
authors.forename: Padmanabhan
authors.initials: P
authors.lastname: Anbazhagan
authors.affiliation: Biocenter Oulu and Department of Biochemistry, University of Oulu, Oulu, FI-90014 Finland.
authors.forename: André H
authors.initials: AH
authors.lastname: Juffer
authors.affiliation: From the Department of Advanced Bioscience, Kinki University, Nara 631-8505 Japan,. Electronic address: fukamizo@nara.kindai.ac.jp.
authors.forename: Tamo
authors.initials: T
authors.lastname: Fukamizo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D048271:Chitosan / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D056507:Paenibacillus / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase
keywords: Carbohydrate-binding Protein; Docking; Isothermal Titration Calorimetry; NMR; Oligosaccharide