The catalytic mechanism of indole-3-glycerol phosphate syntase:: Crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue, substrate, and product
The catalytic mechanism of indole-3-glycerol phosphate syntase:: Crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue, substrate, and product
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DOI：
10.1016/s0022-2836(02)00378-9
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发表时间：
2002-06-07
影响因子：
5.6
通讯作者：
Kirschner, K
中科院分区：
文献类型：
--
作者：
Hennig, M;Darimont, BD;Kirschner, K
吲哚甘油磷酸合酶催化 N-烷基化邻氨基苯甲酸酯闭环生成 3-烷基吲哚衍生物，该反应需要路易斯酸在体外催化。在这里，我们通过 X 射线晶体学研究了来自硫磺硫化叶菌的超热稳定酶与底物 1-(邻羧基苯氨基) 1-脱氧核酮糖 5-磷酸、底物类似物和产物吲哚-3-甘油磷酸的复合物的酶反应机制。底物和底物类似物以类似的延伸构象结合到活性位点上，该构象位于先前鉴定的磷酸盐结合位点和邻氨基苯甲酸盐部分的疏水口袋之间。这种结合模式效率低下，因为要连接的碳原子距离太远。结合产物的吲哚环位于与底物的邻氨基苯甲酸盐部分相邻的第二疏水袋中。尽管底物的疏水部分在催化过程中从一个疏水口袋移动到另一个疏水口袋，但磷酸丙糖部分仍然刚性地结合到同一组氢键残基上。同时，催化上重要的残基 Lys53、Lys110 和 Glu159 与发生共价变化的配体原子保持有利的距离。根据这些数据，将两种假定的催化中间体的结构建模为活性位点。这种新的结构信息和建模研究为酶催化吲哚合成的机制提供了进一步的见解。 Lys110 的带电 e-氨基是一般酸，Glu159 的羧酸根是一般碱。 Lys53 通过与其邻氨基苯甲酸盐部分的羧酸盐基团形成盐桥，引导底物在催化过程中经历构象转变。 (C) 2002 Elsevier Science Ltd. 保留所有权利。
Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged e-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety. (C) 2002 Elsevier Science Ltd. All rights reserved.