Publication Date:
2014-09-04
Description:
Tri- N -acetylchitotriosyl moranoline, (GlcNAc) 3 -M, was previously shown to strongly inhibit lysozyme (Ogata M, Umemoto N, Ohnuma T, Numata T, Suzuki A, Usui T, Fukamizo T. 2013. A novel transition-state analogue for lysozyme, 4-O-β-tri-Nacetylchitotriosyl moranoline, provided evidence supporting the covalent glycosyl-enzyme intermediate. J Biol Chem . 288:6072–6082). The findings prompted us to examine the interaction of di- N -acetylchitobiosyl moranoline, (GlcNAc) 2 -M, with a family GH19 chitinase from moss, Bryum coronatum ( Bc Chi19A). Thermal unfolding experiments using Bc Chi19A and the catalytic acid-deficient mutant ( Bc Chi19A-E61A) revealed that the transition temperature ( T m ) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc) 2 -M, while the chitin dimer, (GlcNAc) 2 , elevated T m only by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc) 3 and (GlcNAc) 2 -M with Bc Chi19A-E61A were determined to be –5.2 and –6.6 kcal/mol, respectively, while (GlcNAc) 2 was found to interact with Bc Chi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using 15 N-labeled Bc Chi19A and Bc Chi19A-E61A revealed that both (GlcNAc) 2 and (GlcNAc) 2 -M interact with the region surrounding the catalytic center of the enzyme and that the interaction of (GlcNAc) 2 -M is markedly stronger than that of (GlcNAc) 2 for both enzymes. However, (GlcNAc) 2 -M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed by Bc Chi19A (IC 50 = 130–620 μM). A molecular dynamics simulation of BcChi19A in complex with (GlcNAc) 2 -M revealed that the complex is quite stable and the binding mode does not significantly change during the simulation. The moranoline moiety of (GlcNAc) 2 -M did not fit into the catalytic cleft (subsite –1) but was rather in contact with subsite +1. This situation may result in the moderate inhibition toward the Bc Chi19A-catalyzed hydrolysis.
Print ISSN:
0959-6658
Electronic ISSN:
1460-2423
Topics:
Biology
,
Medicine
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