Kinetic and structural characterization of a heterohexamer 4-oxalocrotonate tautomerase from Chloroflexus aurantiacus J-10-fl: implications for functional and structural diversity in the tautomerase superfamily .
Kinetic and structural characterization of a heterohexamer 4-oxalocrotonate tautomerase from Chloroflexus aurantiacus J-10-fl: implications for functional and structural diversity in the tautomerase superfamily .
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DOI：
10.1021/bi100502z
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发表时间：
2010-06-22
期刊：
影响因子：
2.9
通讯作者：
Pegan, Scott D.
中科院分区：
文献类型：
--
作者：
Burks, Elizabeth A.;Fleming, Christopher D.;Mesecar, Andrew D.;Whitman, Christian P.;Pegan, Scott D.
4-草酰基-4-羟甲基丙酸互变异构酶(4-OT)同工酶在细菌利用芳香烃作为唯一碳源中起着重要作用。这些酶催化2-羟基-2，4-己二烯二酸酯(或2-羟基己二酸)转化为2-氧代-3-己二烯二酸酯，其中Pro-1起普通碱的作用，并将质子从底物的2-羟基运送到产物的C-5位。4-OT是恶臭假单胞菌Mt-2的同源六聚体，是研究最广泛的4-OT同工酶，也是互变构酶超家族的创始成员。对五个嗜热细菌基因组的搜索发现，每个基因组中都有一个编码的氨基酸序列，被注释为互变构酶样蛋白，但缺少Pro-1。然而，附近的一个序列含有Pro-1，但该序列并未被注释为互变擦除酶样蛋白。为了鉴定这组蛋白，我们克隆了橙色绿僵菌J-10-fl的两个基因，并表达了相应的蛋白。动力学、生化和X-射线结构分析表明，两个表达的蛋白质形成了一个由三个αβ二聚体组成的功能性杂六角体4-OT(HH4-OT)。与恶臭假单胞菌的酶一样，hh4-OT需要氨基末端的脯氨酸和两个精氨酸来将2-羟基肉豆酸转化为产物，这意味着一个类似的机制。与4-OT相比，hH4-OT没有表现出另一个互变构酶超家族成员--异六聚体反式-3-氯丙烯酸脱卤酶(CAAD)的低水平活性。对HH4-OT的表征使相关酶的功能分配成为可能，突出了β-α-β构建块可以组装成活性酶的不同方式，并为进一步了解4-OT中低水平CAAD活性的分子基础提供了进一步的见解。
4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of substrate to the C-5 position of product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. In order to characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the corresponding proteins expressed. Kinetic, biochemical, and X-ray structural analysis show that the two expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three αβ dimers. Like the P. putida enzyme, the hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to product, implicating an analogous mechanism. In contrast to 4-OT, the hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase (CaaD). Characterization of the hh4-OT enables functional assignment of the related enzymes, highlights the diverse ways the β–α–β building block can be assembled into an active enzyme, and provides further insight into the molecular basis of the low level CaaD activity in 4-OT.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
Almrud, JJ;Kern, AD;Whitman, CP
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Whitman, CP
影响因子：
2.9
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Czerwinski, RM;Harris, TK;Whitman, CP
通讯作者：
Whitman, CP
影响因子：
2.9
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Poelarends, Gerrit J.;Almrud, Jeffrey J.;Whitman, Christian P.
通讯作者：
Whitman, Christian P.
影响因子：
2.9
作者：
Almrud, JJ;Poelarends, GJ;Whitman, CP
通讯作者：
Whitman, CP