title: Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii.
abstract: We report here the role and mechanism of specificity of a family 32 carbohydrate binding module (CBM32) of a glycoside hydrolase family 8 chitosanase from Paenibacillus elgii (PeCsn). Both the activity and mode of action of PeCsn toward soluble chitosan polymers were not different with/without the CBM32 domain of P. elgii (PeCBM32). The decreased activity of PeCsn without PeCBM32 on chitosan powder suggested that PeCBM32 increases the relative concentration of enzyme on the substrate and thereby enhanced enzymatic activity. PeCBM32 specifically bound to polymeric and oligomeric chitosan and showed very weak binding to chitin and cellulose. In isothermal titration calorimetry, the binding stoichiometry of 2 and 1 for glucosamine monosaccharide (GlcN) and disaccharide (GlcN)2, respectively, was indicative of two binding sites in PeCBM32. A three-dimensional model-guided site-directed mutagenesis and the use of defined disaccharides varying in the pattern of acetylation suggested that the amino groups of chitosan and the polar residues Glu-16 and Glu-38 of PeCBM32 play a crucial role for the observed binding. The specificity of CBM32 has been further elucidated by a generated fusion protein PeCBM32-eGFP that binds to the chitosan exposing endophytic infection structures of Puccinia graminis f. sp. tritici Phylogenetic analysis showed that CBM32s appended to chitosanases are highly conserved across different chitosanase families suggesting their role in chitosan recognition and degradation. We have identified and characterized a chitosan-specific CBM32 useful for in situ staining of chitosans in the fungal cell wall during plant-fungus interaction.
authors.affiliation: From the Department of Plant Sciences, School of Life Sciences.
authors.forename: Subha Narayan
authors.initials: SN
authors.lastname: Das
authors.affiliation: the Institute of Plant Biology and Biotechnology, Westphalian Wilhelms-University of Münster, 48143 Münster, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Wagenknecht
authors.affiliation: Department of Biochemistry, and.
authors.forename: Pavan Kumar
authors.initials: PK
authors.lastname: Nareddy
authors.affiliation: From the Department of Plant Sciences, School of Life Sciences.
authors.forename: Bhoopal
authors.initials: B
authors.lastname: Bhuvanachandra
authors.affiliation: School of Chemistry, University of Hyderabad, 500046-Hyderabad, India and.
authors.forename: Ramana
authors.initials: R
authors.lastname: Niddana
authors.affiliation: School of Chemistry, University of Hyderabad, 500046-Hyderabad, India and.
authors.forename: Rengarajan
authors.initials: R
authors.lastname: Balamurugan
authors.affiliation: School of Chemistry, University of Hyderabad, 500046-Hyderabad, India and.
authors.forename: Musti J
authors.initials: MJ
authors.lastname: Swamy
authors.affiliation: the Institute of Plant Biology and Biotechnology, Westphalian Wilhelms-University of Münster, 48143 Münster, Germany.
authors.forename: Bruno M
authors.initials: BM
authors.lastname: Moerschbacher
authors.affiliation: From the Department of Plant Sciences, School of Life Sciences, podilerao@gmail.com.
authors.forename: Appa Rao
authors.initials: AR
authors.lastname: Podile
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048271:Chitosan / Q000737:chemistry* / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D005944:Glucosamine / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D000072417:Protein Domains
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D048271:Chitosan; D006026:Glycoside Hydrolases; D005944:Glucosamine
keywords: Paenibacillus elgii; carbohydrate-binding protein; chitin; chitosan; chitosanase; glycoside hydrolase; in situ staining; isothermal titration calorimetry (ITC); site-directed mutagenesis