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

Shoko Shinya, Shigenori Nishimura, Yoshihito Kitaoku, Tomoyuki Numata, Hisashi Kimoto, Hideo Kusaoke, Takayuki Ohnuma, Tamo Fukamizo

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosanbinding 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-15 N-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 nonreducing end at the binding site. Glu14 of DD2 appeared to make an electrostatic interaction with the amino group of the nonreducing 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.

Original languageEnglish
Pages (from-to)1085-1095
Number of pages11
JournalBiochemical Journal
Volume473
Issue number8
DOIs
Publication statusPublished - Apr 15 2016

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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