In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS–phosphoribosylanthranilate isomerase fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at an even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, of which seven contain ligand and one displays a previously unobserved conformation closer to the reactive state. One of the few nonconserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that despite IGPS's classification as a carboxy-lyase (i.e. decarboxylase), decarboxylation is not a completely essential step in its catalysis.

中文翻译：

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铜绿假单胞菌的吲哚-3-甘油磷酸合酶的结构和动力学-脱羧对于吲哚的形成不是必需的。

在色氨酸生物合成中，吲哚-3-甘油磷酸合酶（IGPS）催化的反应从缩合步骤开始，在该步骤中，底物的羧基化苯基进行亲核攻击，形成吲哚的吡咯环，然后进行脱羧反应，恢复苯基的芳香性。铜绿假单胞菌的IGPS在所有特征性IGPS酶中具有最高的周转率，为测试脱羧步骤的必要性提供了一个出色的模型系统。自1960年代以来，根据对大肠埃希氏菌IGPS-磷酸核糖基氰基氨基甲酸酯异构酶融合蛋白的研究，这一步骤被认为是机械上必不可少的。在这里，我们介绍了铜绿假单胞菌IGPS与降低的CdRP（一种非反应性底物类似物）形成复合物的晶体结构，并使用灵敏的不连续测定法，我们证明了对脱羧化底物1-（苯氨基）-1-脱氧核糖-5-磷酸的混合活性较弱，与天然底物相比，IGP的形成速率低约1000倍。我们还表明，大肠杆菌IGPS甚至可以以更低的速率从脱羧的底物中产生IGP。我们的铜绿假单胞菌IGPS的结构在不对称单元中有8个分子，其中7个含有配体，一个分子显示出以前未观察到的构象，更接近反应态。铜绿假单胞菌IGPS中的少数几个非保守活性位点残基之一Phe201已通过诱变证明对于该酶在两个底物上的更高转化都是重要的。我们的结果表明，尽管IGPS被归类为羧裂酶（即脱羧酶），