Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli
Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli
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DOI：
10.1074/jbc.m808186200
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发表时间：
2009-02-06
影响因子：
4.8
通讯作者：
Yakunin, Alexander F.
中科院分区：
文献类型：
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作者：
Brown, Greg;Singer, Alexander;Yakunin, Alexander F.
糖异生是一种重要的代谢途径，它从非碳水化合物前体（例如有机酸、脂肪酸、氨基酸或甘油）产生葡萄糖。果糖-1,6-二磷酸酶是糖异生的关键酶，存在于所有生物体中，并且已鉴定出五种不同类别的这些酶。在这里，我们证明大肠杆菌有两种 II 类果糖-1,6-双磷酸酶：GlpX 和 YggF，它们表现出不同的催化特性。我们展示了 II 类果糖 1,6-二磷酸酶 (GlpX) 的第一个晶体结构，该酶在游离状态和与底物（果糖 1,6-二磷酸）或抑制剂（磷酸盐）的复合物中测定。无配体 GlpX 的晶体结构显示出紧凑的球状形状，具有两个 α/β 夹心结构域。 GlpX的核心折叠结构与Li+敏感磷酸酶的核心折叠结构相似，这意味着它们具有共同的进化起源和催化机制。 GlpX 与果糖 1,6-二磷酸复合物的结构表明，活性位点位于两个结构域之间，并容纳几个协调两个金属离子和底物的保守残基。第三金属离子与底物的磷酸盐6结合。无机磷酸盐强烈抑制 GlpX 和 YggF 的活性，GlpX 与磷酸盐复合物的晶体结构表明抑制剂分子与活性位点结合。 GlpX 的丙氨酸替代诱变鉴定出 12 个对活性重要的保守残基，并表明 Thr(90) 是主要催化残基。我们的数据提供了对 GlpX 和其他 II 类果糖 1,6-二磷酸酶的底物特异性和催化作用的分子机制的深入了解。
Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two alpha/beta-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr(90) is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.