Interaction of di-N-acetylchitobiosyl moranoline with a family GH19 chitinase from moss, Bryum coronatum.
Interaction of di-N-acetylchitobiosyl moranoline with a family GH19 chitinase from moss, Bryum coronatum.
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二-N-乙酰基壳二糖基吗啉与来自苔藓、Bryum coronatum 的 GH19 几丁质酶家族的相互作用。
作者：
Shinya;S.;Urasaki;A.;Ohnuma;T.;Taira;T.;Suzuki;A.;Ogata;M.;Usui;T.;Lampela;O.;Juffer;AH.;Fukamizo;T.
三-N-乙酰基壳三糖基吗啉啉（GlcNAc）3-M先前显示强烈抑制溶菌酶（Ogata M，Umemoto N，Ohnuma T，Numata T，Suzuki A，Jiami T，Jiamizo T. 2013.溶菌酶的一种新的过渡态类似物4-O-β-tri-N-acetylchitotriosyl moranoline提供了支持共价糖基-酶中间体的证据。J Biol Chem.288：6072-6082）。这一发现促使我们研究二-N-乙酰壳二糖基moranoline（GlcNAc）2-M与来自藓类植物冠状真藓（Bryum coronatum）的家族GH 19几丁质酶（BcChi 19 A）的相互作用。使用BcChi 19 A和催化酸缺陷突变体（BcChi 19 A-E61 A）的热展开实验显示，在加入（GlcNAc）2-M后，转变温度（Tm）分别升高了4.3和5.8°C，而几丁质二聚体（GlcNAc）2使Tm分别升高了1.0和1.4°C。通过等温滴定量热法，（GlcNAc）3和（GlcNAc）2-M与BcChi 19 A-E61 A相互作用的结合自由能变化分别为−5.2和−6.6 kcal/mol，而（GlcNAc）2与BcChi 19 A-E61 A相互作用的亲和力明显较低。用~（15）N标记的BcChi 19 A和BcChi 19 A-E61 A进行的核磁共振滴定实验表明，（GlcNAc）_2和（GlcNAc）_2-M都与酶的催化中心周围区域相互作用，且（GlcNAc）_2-M的相互作用明显强于（GlcNAc）_2。但（GlcNAc）2-M对BcChi 19 A催化的甲壳低聚糖水解反应有一定的抑制作用（IC_（50）= 130-620 μM）。BcChi 19 A与（GlcNAc）2-M复合物的分子动力学模拟显示，该复合物相当稳定，并且在模拟过程中结合模式没有显著改变。（GlcNAc）2-M的吗啉部分不适合催化裂缝（亚位点-1），而是与亚位点+1接触。这种情况可能导致对BcChi 19 A催化的水解的适度抑制。
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(BcChi19A). Thermal unfolding experiments usingBcChi19A 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, elevatedTmonly by 1.0 and 1.4°C, respectively. By means of isothermal titration calorimetry, binding free energy changes for the interactions of (GlcNAc)3and (GlcNAc)2-M withBcChi19A-E61A were determined to be −5.2 and −6.6 kcal/mol, respectively, while (GlcNAc)2was found to interact withBcChi19A-E61A with markedly lower affinity. nuclear magnetic resonance titration experiments using15N-labeledBcChi19A andBcChi19A-E61A revealed that both (GlcNAc)2and (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)2for both enzymes. However, (GlcNAc)2-M was found to moderately inhibit the hydrolytic reaction of chitin oligosaccharides catalyzed byBcChi19A (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 theBcChi19A-catalyzed hydrolysis.