Structural and Kinetic Analysis of Bacillus subtilis N-Acetylglucosaminidase Reveals a Unique Asp-His Dyad Mechanism
Structural and Kinetic Analysis of Bacillus subtilis N-Acetylglucosaminidase Reveals a Unique Asp-His Dyad Mechanism
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DOI：
10.1074/jbc.m110.131037
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发表时间：
2010-11-12
影响因子：
4.8
通讯作者：
Mayer, Christoph
中科院分区：
文献类型：
--
作者：
Litzinger, Silke;Fischer, Stefanie;Mayer, Christoph
三维结构的枯草芽孢杆菌的NagZ，第一个结构的两个结构域β-N-乙酰氨基葡萄糖苷酶的第3家族的糖苷酶，确定有和没有过渡态模拟抑制剂PUGNAc结合到活性位点，分别在1.84和1.40埃的分辨率。突变体的结构和动力学分析揭示了一个Asp-His二联体参与催化作用：BsNagZ的His（234）作为一般的酸/碱催化剂，并通过Asp（232）氢键合以实现适当的功能。用甘氨酸取代His 234和Asp（232），可使荧光底物4 '-甲基伞形酮基N-乙酰基-β-D-氨基葡萄糖苷的水解速率分别降低1900倍和4500倍，并使活性在碱性范围内不依赖于pH，这与这些残基在酸/碱催化中的作用一致。N-乙酰葡糖胺酶中间体在H234 G突变体中积累，并且在两种突变体中在叠氮化钠存在下形成β-叠氮化物产物。Asp-His二联体在β-N-乙酰氨基葡糖苷酶中是保守的，但在家族3的β-葡糖苷酶中不存在，其携带“经典的”谷氨酸/碱催化剂。大麦外切葡聚糖酶（Exo 1）的酸/碱谷氨酸与BsNagZ二分体的His 234重叠，与后者相反，从酶的第二结构域突出到活性位点。这是第一次报告的Asp-His催化二联体参与水解的糖苷类似的Asp-His-Ser三联体的丝氨酸蛋白酶的功能。我们的研究结果将促进基于机制的抑制剂的发展，选择性靶向家族3 β-N-乙酰氨基葡萄糖苷酶，这是参与细菌细胞壁周转，孢子萌发，和β-内酰胺酶的诱导。
Three-dimensional structures of NagZ of Bacillus subtilis, the first structures of a two-domain beta-N-acetylglucosaminidase of family 3 of glycosidases, were determined with and without the transition state mimicking inhibitor PUGNAc bound to the active site, at 1.84- and 1.40-angstrom resolution, respectively. The structures together with kinetic analyses of mutants revealed an Asp-His dyad involved in catalysis: His(234) of BsNagZ acts as general acid/base catalyst and is hydrogen bonded by Asp(232) for proper function. Replacement of both His234 and Asp(232) with glycine reduced the rate of hydrolysis of the fluorogenic substrate 4'-methylumbelliferyl N-acetyl-beta-D-glucosaminide 1900- and 4500-fold, respectively, and rendered activity pH-independent in the alkaline range consistent with a role of these residues in acid/base catalysis. N-Acetylglucosaminyl enzyme intermediate accumulated in the H234G mutant and beta-azide product was formed in the presence of sodium azide in both mutants. The Asp-His dyad is conserved within beta-N-acetylglucosaminidases but otherwise absent in beta-glycosidases of family 3, which instead carry a "classical" glutamate acid/base catalyst. The acid/base glutamate of Hordeum vulgare exoglucanase (Exo1) superimposes with His234 of the dyad of BsNagZ and, in contrast to the latter, protrudes from a second domain of the enzyme into the active site. This is the first report of an Asp-His catalytic dyad involved in hydrolysis of glycosides resembling in function the Asp-His-Ser triad of serine proteases. Our findings will facilitate the development of mechanism-based inhibitors that selectively target family 3 beta-N-acetylglucosaminidases, which are involved in bacterial cell wall turnover, spore germination, and induction of beta-lactamase.