Amino Groups of Chitosan Are Crucial for Binding to a Family 32 Carbohydrate Binding Module of a Chitosanase from Paenibacillus elgii.
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.
['Das|Subha Narayan|SN|', 'Wagenknecht|Martin|M|', 'Nareddy|Pavan Kumar|PK|', 'Bhuvanachandra|Bhoopal|B|', 'Niddana|Ramana|R|', 'Balamurugan|Rengarajan|R|', 'Swamy|Musti J|MJ|', 'Moerschbacher|Bruno M|BM|', 'Podile|Appa Rao|AR|']
D001426:Bacterial Proteins D048271:Chitosan D004187:Disaccharides D005944:Glucosamine D006026:Glycoside Hydrolases D008958:Models, Molecular D016297:Mutagenesis, Site-Directed D056507:Paenibacillus D011485:Protein Binding D000072417:Protein Domains
chitosan chitosanase Paenibacillus elgii glycoside hydrolase chitin site-directed mutagenesis in situ staining carbohydrate-binding protein isothermal titration calorimetry (ITC)