Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase
Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase
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DOI：
10.1093/emboj/cdg403
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发表时间：
2003-08-15
期刊：
影响因子：
11.4
通讯作者：
Grütter, MG
中科院分区：
文献类型：
--
作者：
Capitani, G;De Biase, D;Grütter, MG
谷氨酸脱羧酶是一种维生素B6依赖性酶，其催化谷氨酸脱羧为γ-氨基丁酸。在大肠杆菌中，谷氨酸脱羧酶（GadB）（一种330 kDa的六聚体）的表达被诱导以在酸性条件下维持生理pH，就像在通过胃到达肠的途中的那些。GadB与反向转运蛋白GadC一起构成了gad耐酸性系统，该系统赋予细菌在强酸性环境中存活至少2 h的能力。GadB经历了一个pH依赖的构象变化，并表现出在低pH值的活性最佳。我们确定了GadB的晶体结构在酸性和中性pH值。他们揭示了分子的构象变化的细节和结构基础的酸性pH值最佳。我们证明，该酶是专门定位在细胞质中在中性pH值，但被招募到膜时，pH值福尔斯下降。我们表明，基于结构的定点突变，在酸性pH值的蛋白质的N-末端形成的三螺旋束是这种行为的主要决定因素。
Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.