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首页> 外文期刊>Biochemistry >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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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

机译：动力学和结构表征的异六聚体4-氧杂巴豆酸酯互变异构酶从Chloroflexus aurantiacus J-10-fl：互变异构酶超家族中功能和结构多样性的影响。

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4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilizationnof aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-nhexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general basenand shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, anhomohexamer fromPseudomonas putidamt-2, is themost extensively studied 4-OT isozyme and the foundingnmember of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a codednamino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1.nHowever, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. Toncharacterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and thencorresponding proteins were expressed.Kinetic, biochemical, andX-ray structural analyses show that the twonexpressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three Rβ dimers. Like thenP. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion ofn2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT doesnnot exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-nchloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of thenrelated enzymes, highlights the diverse ways the β-R-β building block can be assembled into an activenenzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.
机译：4-羟基巴豆酸互变异构酶（4-OT）同工酶在细菌利用芳香烃作为唯一碳源方面发挥着重要作用。这些酶催化2-羟基-2,4-己二烯二酸酯（或2-羟基粘康酸酯）向2-oxo-3-hexenedioate的转化，其中Pro-1用作一般碱基，并从质子的2-羟基基团转移质子。底物到产品的C-5位置。 4-OT是恶臭假单胞菌（Pseudomonas putidamt-2）的六聚体，是研究最广泛的4-OT同工酶，是互变异构酶超家族的创始成员。对五个嗜热细菌基因组进行搜索后，发现每个基因组中的一个编码氨基酸序列都被标注为互变异构酶样蛋白但缺少Pro-1.n然而，附近的序列具有Pro-1，但该序列并未被标注为互变异构酶-像蛋白质。为了表征这组蛋白质，克隆了来自Chloroflexus aurantiacus J-10-fl的两个基因，然后表达了相应的蛋白质。动力学，生化和X射线结构分析表明，这两个被压下的蛋白质形成了功能性异六聚体4-OT（hh4-OT ），由三个Rβ二聚体组成。就像thenP。腐殖酸酶hh4-OT需要氨基末端的脯氨酸和两个精氨酸才能将n2-羟基粘康酸酯转化为产物，这暗示了类似的机制。与4-OT相比，hh4-OT没有表现出另一个互变异构酶超家族成员，异六聚体反式3-n氯丙烯酸脱卤素酶（CaaD）的低水平活性。 hh4-OT的表征可实现相关酶的功能分配，突出显示可将β-R-β结构单元组装成活性酶的多种方式，并可进一步洞察4-OT中低水平CaaD活性的分子基础。

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  • 来源
    《Biochemistry》 |2010年第24期|p.5016-5027|共12页
  • 作者

    Elizabeth A. Burks Christopher D. Fleming Andrew D. Mesecar Christian P. Whitman and Scott D. Pegan;

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    Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712,) Center for PharmaceuticalBiotechnology (MC 870), College of Pharmacy, University of Illinois, 900 South Ashland Avenue, Chicago, Illinois 60607-7173, and^Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208-0183;

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