Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris
Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris
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DOI：
10.1128/jb.183.13.4061-4070.2001
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发表时间：
2001-07-01
影响因子：
3.2
通讯作者：
Jensen, RA
中科院分区：
文献类型：
--
作者：
Gosset, G;Bonner, CA;Jensen, RA
进行芳香族氨基酸生物合成的初始反应2-酮-3-脱氧-D-阿拉伯庚酮糖酸7-磷酸（DAHP）合成的酶以两种不同的同源性类别存在。三种经典的大肠杆菌旁系同源物是AroA（I）蛋白，但细菌的许多成员具有AroA（II）类酶，有时与AroA（I）蛋白组合。迄今为止，AroA（II）DAHP合成已被证明专门用于次级代谢（例如，形成安莎霉素抗生素或吩嗪色素）。相反，在这里，我们表明，黄单胞菌AroA（II）蛋白的功能作为唯一的DAHP合成酶支持芳香族氨基酸的生物合成。X. campestris AroA（II）基因在E. coli中进行功能互补，表达了对应于两种可能翻译起始点的基因。我们开发了一种1天的部分纯化方法（> 99%）的不稳定蛋白。重组AroA（II）蛋白被发现是受变构模式的顺序反馈抑制，其中分支酸是主要的变构效应。发现L-色氨酸是一种次要的反馈抑制剂。一个111个氨基酸的N-末端区域可能位于周质中，因为一个可能的内膜跨区域的预测。与高等植物的叶绿体定位的AroA（II）不同，X.油菜AroA（II）不被二硫醇滞后激活。与植物AroA（II）蛋白相比，还观察到二价金属活化的差异。系统发育树分析表明，AroA（II）起源于细菌结构域，它似乎可能是高等植物质体获得AroA（II）从一个革兰氏阴性细菌通过内共生。副艇长campestris AroA（II）蛋白被认为是一种类似物置换的情况，由此祖先aroA（I）种类被丢弃，aroA（II）置换提供了变构控制的替代模式。AroA，，蛋白质可以分为三个亚群：一个是包含植物酶的大的中心组，另一个是来自X. campestris，一个由保守的KPRS基序附近的三个残基缺失定义，另一个在下游具有更大的缺失。
Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) syntheses, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA(I) proteins, but many members of the Bacteria possess the AroA(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA(II) DAHP syntheses until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (> 99%) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA(II) of higher plants, X. campestris AroA(II) was not hysteretically activated by dithiols. Compared to plant AroA(II) proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA(II) originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. campestris AroA(II) protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA(I) species was discarded, with the aroA(II) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA,, proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.