The binding mode of the methylkasugaminide moiety was almost the same among the different GH18 chitinases whereas the binding mode of the D-inositol moiety differed. to the substrate. (GS115. was expressed in BL21 (DE3). All the proteins were purified from the culture medium by immobilized metal affinity chromatography (IMAC) as described previously (Chen et al., 2014). The purities of the target proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. Inhibitory Activity Determination Briefly, the reaction mixtures used for inhibitor screening had a final assay volume of 100?L. 20?nM enzyme was incubated with 10?L substrate [0.2?M MU–(GlcNAc)2] in 20?mM sodium phosphate buffer (pH 6.0 for (Fuchs et al., 1986), (Liu et al., 2017), and (Fusetti et al., 2002; Bussink et al., 2008) were studied. Inhibition kinetics demonstrated that kasugamycin inhibits all of the tested GH18 chitinases in a competitive mode (Figure 1B; Supplementary Figure S1) with em K /em i values varying from 0.25 to 29.00?M (Table 1). TABLE 1 Inhibitory activities and binding affinities of the compounds toward different GH18 chitinases. thead valign=”top” th align=”left” rowspan=”1″ colspan=”1″ Organism /th th align=”center” rowspan=”1″ colspan=”1″ Name /th th align=”center” rowspan=”1″ colspan=”1″ em K /em i (M) /th th align=”center” rowspan=”1″ colspan=”1″ em K /em d (M) /th /thead Human em Hs /em Cht0.25 (1.62) a 0.92AMCase6.2715.84Insect em Butylparaben Of /em ChtI0.473.96 em Of /em Chi-h2.711.5Bacterium em Sm /em ChiA29.0034.11 Open in a separate window aThe em K /em i of kasugamycin against em Hs /em Cht in the buffer with 1.0?M NaCl. Since the SBCs of GH18 chitinases usually contain several solvent-exposed tryptophan residues, tryptophan fluorescence quenching spectroscopy was used to determine the binding affinity of kasugamycin to GH18 chitinases. As shown in Figure 1C and Supplementary Figure S2, kasugamycin quenched the native tryptophan fluorescence of GH18 chitinases in a dose-dependent mode. The equilibrium dissociation constant ( em K /em d) values of kasugamycin to GH18 chitinases varied from 0.92 to 34.11?M (Table 1). The tendency of the em K /em d values is in good accordance with that of the em K /em i values, although the values are not identical. To further understand the inhibitory mechanism, kasugamycin was docked into the crystal structure of em Hs /em Cht (Fusetti et al., 2002), which has the highest affinity toward kasugamycin. Although there is little structural similarity between kasugamycin and CHOS, kasugamycin bound the SBC of em Hs /em Cht in a similar mode as (GlcNAc)2 by forming CH- interactions with the indole group of Trp31 and Trp358 (Figure 2A). The methylkasugaminide moiety occupied the C1 subsite of the SBC and formed hydrogen bonds with surrounding residues including Glu140, Tyr141 and Asp213. The D-inositol moiety of kasugamycin occupied the C2 subsite of the SBC and formed a hydrogen bond with Asn100. Since the amino group of kasugamycin and the carboxyl group of Asp138 (one of the catalytic triad residues) have opposite charges at pH 6.0, we hypothesized that the strong electrostatic interaction between them was a driving force for the inhibitory activity of kasugamycin against GH18 chitinases. To prove this hypothesis, we determined the em K /em i value of kasugamycin against em Hs /em Cht in a buffer containing 1.0?M NaCl to weaken the electrostatic interaction. Under these conditions, the em K /em i value of kasugamycin against em Hs /em Cht increased 6-fold to 1 1.62?M (Table 1; Supplementary Figure S3), demonstrating the importance of this electrostatic interaction in the binding affinity of kasugamycin to GH18 chitinases. Most of residues involved in binding were key residues for chitinase catalysis. Residues Asp138 and Glu140 are responsible for glycosidic bond breaking. Residue Asp213 is involved in catalysis by stabilizing the ?1 sugar in its distorted conformation (Synstad et al., 2004; Chen et al., 2020). Mutation of these residues in em Sm /em ChiB yielded greatly reduction in catalytic activity (Synstad et al., 2004). Butylparaben Kasugamycin was first reported as a bacterial protein synthesis inhibitor, and the binding mechanism of kasugamycin to the 30S subunit of the bacterial ribosome has been studied by X-ray crystallography (Schluenzen et al., 2006). In this structure, kasugamycin binds the 16S ribosomal RNA within the messenger RNA channel. The electrostatic interaction formed between the amino group of Rabbit Polyclonal to EPHA3/4/5 (phospho-Tyr779/833) kasugamycin and the backbone phosphate group of G1483 was also important.The tendency of the em K /em d values is in good accordance with that of the em K /em i values, although the values are not identical. To further understand the inhibitory mechanism, kasugamycin was docked into the crystal structure of em Hs /em Cht (Fusetti et al., 2002), which has the highest affinity toward kasugamycin. The purities of the target proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining. Inhibitory Activity Determination Briefly, the reaction mixtures used for inhibitor screening had a final assay volume of 100?L. 20?nM enzyme was incubated with 10?L substrate [0.2?M MU–(GlcNAc)2] in 20?mM sodium phosphate buffer (pH 6.0 for (Fuchs et al., 1986), (Liu et al., 2017), and (Fusetti et al., 2002; Bussink et al., 2008) were studied. Inhibition kinetics demonstrated that kasugamycin inhibits all of the tested GH18 chitinases in a competitive mode (Figure 1B; Supplementary Figure S1) with em K /em i values varying from 0.25 to 29.00?M (Table 1). TABLE 1 Inhibitory activities and binding affinities of the compounds toward different GH18 chitinases. thead valign=”top” th align=”left” rowspan=”1″ colspan=”1″ Organism /th th align=”center” rowspan=”1″ colspan=”1″ Name /th th align=”center” rowspan=”1″ colspan=”1″ em K /em i (M) /th th align=”center” rowspan=”1″ colspan=”1″ em K /em d (M) /th /thead Human em Hs /em Cht0.25 (1.62) a 0.92AMCase6.2715.84Insect em Of /em ChtI0.473.96 em Of /em Chi-h2.711.5Bacterium em Sm /em ChiA29.0034.11 Open in a separate window aThe em K /em i of kasugamycin against em Hs /em Cht in the buffer with 1.0?M NaCl. Since the SBCs of GH18 chitinases usually contain several solvent-exposed tryptophan residues, tryptophan fluorescence quenching spectroscopy was used to determine the binding affinity of kasugamycin to GH18 chitinases. As shown in Figure 1C and Supplementary Figure S2, kasugamycin quenched the native tryptophan fluorescence of GH18 chitinases in a dose-dependent mode. The equilibrium dissociation constant ( em K /em d) values of kasugamycin to GH18 chitinases varied from 0.92 to 34.11?M (Table 1). The tendency of the em K /em d values is in good accordance with that of the em K /em i values, although the values are not identical. To further understand the inhibitory mechanism, kasugamycin was docked into the crystal structure of em Hs /em Cht (Fusetti et al., 2002), which has the highest affinity toward kasugamycin. Although there is little structural similarity between kasugamycin and CHOS, kasugamycin bound the SBC of em Hs /em Cht in a similar mode as (GlcNAc)2 by forming CH- interactions with the indole group of Trp31 and Trp358 (Figure 2A). The methylkasugaminide moiety occupied the C1 subsite of the SBC and formed hydrogen bonds with surrounding residues including Glu140, Tyr141 and Asp213. The D-inositol moiety of kasugamycin occupied the C2 subsite of the SBC and formed a hydrogen bond with Asn100. Since the amino group of kasugamycin and the carboxyl group of Asp138 (one of the catalytic triad residues) have opposite charges at pH 6.0, we hypothesized that the strong electrostatic interaction between them was a driving force for the inhibitory activity of kasugamycin against GH18 chitinases. To prove this hypothesis, we determined the em K /em i value of kasugamycin against em Hs /em Cht in a buffer containing 1.0?M NaCl to weaken the electrostatic interaction. Under these conditions, the em K /em i value of kasugamycin against em Hs /em Cht increased 6-fold to 1 1.62?M (Table 1; Supplementary Figure S3), demonstrating the importance of this electrostatic interaction in the binding affinity of kasugamycin to GH18 chitinases. Most of Butylparaben residues involved in binding were key residues for chitinase catalysis. Residues Asp138 and Glu140 are responsible for glycosidic bond breaking. Residue Asp213 is involved in catalysis by stabilizing the ?1 sugar in its distorted conformation (Synstad et al., 2004; Chen et al., 2020). Mutation of these residues in em Sm /em ChiB yielded greatly reduction in catalytic activity (Synstad et al., 2004). Kasugamycin was first reported as a bacterial protein synthesis inhibitor, and the binding mechanism of kasugamycin to the 30S subunit of the bacterial ribosome has been studied by X-ray crystallography (Schluenzen et al., 2006). In this structure, kasugamycin binds the 16S ribosomal RNA.