Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp. IK-5 chitosanase/glucanase

An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a β-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in ¹H-¹⁵N-HSQC resonances, the chitosan tetramer (GlcN)₄ was found to bind to the loop region extruded from the core β-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)₃, in which the bound (GlcN)₃ stood upright on its non-reducing end at the binding site. Glu¹⁴ of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg³¹, Tyr³⁶ and Glu⁶¹ formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr³⁶ of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr³⁶ to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.