Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa -: Evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH
Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa -: Evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH
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DOI：
10.1074/jbc.m107249200
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发表时间：
2001-11-16
影响因子：
4.8
通讯作者：
Amrhein, N
中科院分区：
文献类型：
--
作者：
Kitzing, K;Macheroux, P;Amrhein, N
choris酸合成酶催化5-烯醇丙酮莽草酸3-磷酸中磷酸基和C-(6proR)氢的抗1,4消除，生成choris酸，这是芳香族氨基酸生物合成的主要组成部分。该酶对还原的FMN有绝对的要求，在真菌choris酸合酶的情况下，它是由内在的FMN提供的：NADPH氧化还原酶活性，即这些酶具有额外的催化活性。因此，这些真菌酶被称为“双功能”。从普通面包霉菌粗神经孢子菌中克隆出choris酸合成酶，在大肠杆菌中异种表达，并经三步纯化纯化，达到均匀性。重组IV. crassa choris酸合成酶具有脱失酶活性，即以NADPH为代价催化氧化FMN的还原。使用NADPH作为还原剂，在单次和多次转换条件下观察到一个还原的Ravin中间体，其光谱特征与单功能的choris酸合酶相似，从而证明该中间体是choris酸合酶催化反应的共同产物。此外，在氧气存在下的多次转换实验提供了NADPH结合在底物（5-烯醇丙酮酰石草酸3-磷酸）结合位点内或附近的证据，这表明NADPH与双功能choris酸合成酶的结合嵌入在一般蛋白质结构中，并且不需要特殊的NADPH结合域来产生内在的氧化还原酶活性。
Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN: NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant IV. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced Ravin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.