Fully deacetylated chitooligosaccharides act as efficient glycoside hydrolase family 18 chitinase inhibitors

Abstract

Small molecule inhibitors against chitinases have potential applications as pesticides, fungicides, and antiasthmatics. Here, we report that a series of fully deacetylated chitooligosaccharides (GlcN)2-7 can act as inhibitors against the insect chitinase OfChtI, the human chitinase HsCht, and the bacterial chitinases SmChiA and SmChiB with IC50 values at micromolar to millimolar levels. The injection of mixed (GlcN)2-7 into the fifth instar larvae of the insect Ostrinia furnacalis resulted in 85% of the larvae being arrested at the larval stage and death after 10 days, also suggesting that (GlcN)2-7 might inhibit OfChtI in vivo. Crystal structures of the catalytic domain of OfChtI (OfChtI-CAD) complexed with (GlcN)5,6 were obtained at resolutions of 2.0 Å. These structures, together with mutagenesis and thermodynamic analysis, suggested that the inhibition was strongly related to the interaction between the -1 GlcN residue of the inhibitor and the catalytic Glu(148) of the enzyme. Structure-based comparison showed that the fully deacetylated chitooligosaccharides mimic the substrate chitooligosaccharides by binding to the active cleft. This work first reports the inhibitory activity and proposed inhibitory mechanism of fully deacetylated chitooligosaccharides. Because the fully deacetylated chitooligosaccharides can be easily derived from chitin, one of the most abundant materials in nature, this work also provides a platform for developing eco-friendly inhibitors against chitinases.

Keywords: Chitinase; Crystal Structure; Deacetylated Chitooligosaccharides; Enzyme Inhibitor; Glycoside Hydrolase; Insect Molting; Thermodynamics.

FIGURE 1.

Phenotypes observed after the injection of mixed (GlcN)_2–7 into the fifth instar day 5 of the insect O. furnacalis. Three days after injection with mixed (GlcN)_2–7, 60% of the larvae died at the prepupal stage, with the head capsule and the thoracic legs attached to the body (red arrow). Ten days after injection with mixed (GlcN)_2–7, 25% of the larvae died with their body shrunk seriously (red arrow). No malformation was found in water-injected control groups.

FIGURE 2.

A–F, thermograms and binding isotherms with theoretical fits for the binding of (GlcN)_2–7 to OfChtI: (GlcN)₂ (A), (GlcN)₃ (B), (GlcN)₄ (C), (GlcN)₅ (D), (GlcN)₆ (E), and (GlcN)₇ (F). G, free energy changes for (GlcN)_2–7 binding to OfChtI relative to the number of GlcN units in the saccharide chain. (GlcN)₂ and (GlcN)₃ bind to OfChtI at two sites. For (GlcN)₂, S1 is the strong binding site with a low K_d value, and S2 is the weak binding site with a high K_d value. For (GlcN)₃, S1′ is the strong binding site with a low K_d value, and S2′ is the weak binding site with a high K_d value.

FIGURE 3.

Overall structures of OfChtI-CAD complexed with (GlcN)₅ and (GlcN)₆. A and C, surface representations of OfChtI-CAD complexed with (GlcN)₅ (A) and (GlcN)₆ (C). The ligands (GlcN)₅ and (GlcN)₆, the Asn³⁰⁵-linked N-GlcNAc, and the ligand HEPES are shown as sticks with yellow, blue, and green carbon atoms, respectively. The numbers indicate the subsite to which the GlcN residue is bound. B and D, details of the active cleft with the interactions between OfChtI-CAD and (GlcN)₅ (B) or (GlcN)₆ (D). The ligand is represented as a stick with yellow carbon atoms, and the 2F_o − F_c electron density map around the ligand is contoured at the 2.0σ level. The catalytic residues and the amino acids that interact with the ligand are labeled and are shown as sticks with green and blue carbon atoms, respectively. The numbers indicate the subsite to which the GlcN residue is bound. Hydrogen bonds between the enzyme and the ligand are drawn as dashed lines.

FIGURE 4.

A, the superposition of the ligands (GlcN)₅ and (GlcN)₆ in the active cleft of OfChtI-CAD. (GlcN)₅ and (GlcN)₆ are shown as sticks with yellow and cyan carbon atoms, respectively. The catalytic residue Glu¹⁴⁸ in two complex structures is labeled and shown as sticks with green carbon atoms. The numbers indicate the subsite to which the GlcN residue is bound. Hydrogen bonds between Glu¹⁴⁸ and the ligands are drawn as a dashed line. B, superposition of the inhibitor (GlcN)₅ and the substrate (GlcNAc)_2/3 (PDB entry 3WL1) in the substrate-binding cleft of OfChtI-CAD. (GlcN)₅ and (GlcNAc)_2/3 are shown as sticks with yellow and blue carbon atoms, respectively. The catalytic Glu¹⁴⁸ and Phe³⁰⁹ in the structures in complex with (GlcN)₅ and (GlcNAc)_2/3 are labeled and shown as sticks with green and pink carbon atoms, respectively. The numbers indicate the subsite to which the GlcN and GlcNAc residue is bound. Hydrogen bonds between Glu¹⁴⁸ and the ligands are drawn as dashed lines.