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.
Highlights
Carbohydrate binding modules (CBMs)4 are distinct structural folds of a stretch of amino acids within carbohydrateactive enzymes having carbohydrate binding activity
PeCsn appeared as a multidomain protein with an N-terminal GH8 catalytic domain and a C-terminal CBM32 linked by a fibronectin type III (FN3) domain (Fig. 1)
The overall catalytic efficiency of PeCsn, GH8FN3, and GH8 remained the same suggesting no remarkable influence of PeCBM32 on the activity when 0% degree of acetylation (DA) chitosan solution was the substrate (Table 1)
Summary
Carbohydrate binding modules (CBMs) are distinct structural folds of a stretch of amino acids within carbohydrateactive enzymes having carbohydrate binding activity. The members of CBM32 with low sequence identity are known to bind to one type of sugar moiety, suggesting an inability to predict binding specificity and affinity of family 32 CBMs from their primary structure. Often chitosanases have a single catalytic domain with no auxiliary domains, unlike chitinases and cellulases that are frequently associated with CBMs. The mechanism of chitosan binding and degradation by chitosanases, in comparison with chitin and cellulose degradation, is not fully understood due to a lack of well defined chitosan substrates in terms of their degree of acetylation (DA) and pattern of acetylation (PA). The amorphous nature of chitosan suggests that the pattern of chitosan binding by CBMs present in multidomain chitosanases may differ from CBMs that bind and enhance the degradation of crystalline chitin and cellulose. CBM32-2 appeared to be less specific to chitosan (in comparison with CBM32-1) and showed binding to laminarioligosaccharides and cello-oligosaccharides
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