A novel method for chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by the mutant of inverting family GH19 chitinase using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside as a glycosyl donor.

A novel method for the chemo-enzymatic synthesis of chitin oligosaccharide catalyzed by mutants of BcChi-A, an inverting family GH19 chitinase from Bryum coronatum, has been developed using 4,6-dimethoxy-1,3,5-triazin-2-yl α-chitobioside [DMT-α-(GlcNAc)2)] as a donor substrate. Based on the glycosynthase derived from BcChi-A, Glu70, which acts as a catalytic base, and Ser102, which fixes a nucleophilic water molecule, were changed to generate several single and double mutants of BcChi-A, which were employed in synthetic reactions. Among the double mutants tested, E70G/S102G, E70G/S102C and E70G/S102A were found to successfully synthesize chitotetraose [(GlcNAc)4] from DMT-α-(GlcNAc)2 and (GlcNAc)2; however, the single mutants, E70G, S102G, S102C and S102A, did not. Among the mutants, E70G/S102A showed the highest synthetic activity. This is the first report of a glycosynthase that employs a dimethoxytriazine-type glycoside as a donor substrate.

Interaction of di-N-acetylchitobiosyl moranoline with a family GH19 chitinase from moss, Bryum coronatum.

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-ß-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 (BcChi19A). Thermal unfolding experiments using BcChi19A and the catalytic acid-deficient mutant (BcChi19A-E61A) revealed that the transition temperature (Tm) was elevated by 4.3 and 5.8°C, respectively, upon the addition of (GlcNAc)2-M, while the chitin dimer, (GlcNAc)2, elevated Tm 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 BcChi19A-E61A were determined to be -5.2 and -6.6 kcal/mol, respectively, while (GlcNAc)2 was found to interact with BcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using (15)N-labeled BcChi19A and BcChi19A-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 BcChi19A (IC50 = 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 BcChi19A-catalyzed hydrolysis.