Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors

Abstract

Background: Chitinase inhibitors have potential applications as pesticides, fungicides, and antiasthmatics. Results: Fully deacetylated chitooligosaccharides were determined to be GH18 chitinase inhibitors by thermodynamics and crystallography. Conclusion: Crystal structures reveal a competitive inhibition mode. Significance: This work first reports fully deacetylated chitooligosaccharides acting as chitinase inhibitors, perhaps providing a structural basis for developing eco-friendly inhibitors against chitinases.

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

“…Measurements made at various low concentrations of MU-NAG 2 yielded an IC 50 value of 0.32 Ϯ 0.11 mM. This figure is almost an order of magnitude lower than the IC 50 values measured for the inhibition of ChiA by deacetylated NAG 2 (23), indicating a positive effect of acetyl groups on the binding of NAG 2 to ChiA. The hydrolysis of MUL by Cel7A is consistent with Michaelis-Menten kinetics (15,63).…”

“…The mechanisms underlying this phenomenon are not known, although the strong binding of Cel7A to the polymer chain (17,21) or the use of different mechanisms in hydrolysis of low molecular weight and polymeric substrates, may be responsible (18). For ChiA, the hydrolysis of low molecular weight substrates was shown to be inhibited by deacetylated chitobiose, with an IC 50 value of 4.1 mM (23). The pseudotrisaccharide allosamidin binds to ChiA with a K d of 0.17 M (24).…”

Two chitinase 5 genes from Locusta migratoria: Molecular characteristics and functional differentiation

“…Heterologous expression of insect Cht5 has been successfully performed in the Hi5 and Sf9 cell lines and the yeast Pichia pastoris, and the recombinant protein showed high levels of chitinolytic activity (Gopalakrishnan et al, 1995;Shinoda et al, 2001;Zhu et al, 2008c;Wu et al, 2013). The crystal structure of OfCht5 from O. furnacalis has been determined (Chen et al, 2014a), and a series of fully deacetylated chitooligosaccharides (GlcN)2e7 has been demonstrated as inhibitors of OfCht5 (Chen et al, 2014b).…”

Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase

“…SmChiA, SmChiB, and SmChiC from S. marcescens were expressed in Escherichia coli and purified using IMAC as previously described (50). The F232W/F396W double mutant of SmChiA (SmChiA-F232W/F396W) was produced using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's instruction.…”

“…OfChtI and human chitotriosidase (HsCht) were expressed in P. pastoris and purified using IMAC as described previously (50). SmChiA, SmChiB, and SmChiC from S. marcescens were expressed in Escherichia coli and purified using IMAC as previously described (50).…”