分子生物学作业-翻译

分子酶学工程作业

杨柳Identification of a Missing Link in the Evolution of an Enzyme into a

Transcriptional Regulator

Citation: PLoS ONE 2013, 8(3): e57518.

Editor: Frances M. Sladek, Univeristy of California Riverside, United States of America Abstract

The evolution of transcriptional regulators through the recruitment of DNA-binding domains by enzymes is a widely held notion. However, few experimental approaches have directly addressed this hypothesis.Here we report the reconstruction of a plausible pathway for the evolution of an enzyme into a transcriptional regulator. The BzdR protein is the prototype of a subfamily of prokaryotic transcriptional regulators that controls the expression of genes involved in the anaerobic degradation of benzoate. We have shown that BzdR consists of an N-terminal DNA-binding domain connected through a linker to a C-terminal effector-binding domain that shows significant identity to the shikimate kinase (SK). The construction of active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to the Escherichia coli SKI protein strongly supports the notion that an ancestral SK domain could have been involved in the evolutionary origin of BzdR. The loss of the enzymatic activity of the ancestral SK domain was essential for it to evolve as a regulatory domain in the current BzdR protein. This work also supports the view that enzymes precede the emergence of the regulatory systems that may control their expression. Introduction

Regulation of transcription through the action of small molecules that directly bind to a transcription factor is widespread in all life forms. A large number of transcriptional regulators contain a DNA-binding domain fused to an effector-binding domain. Binding of the effector results in a conformational change, which influences the properties of the transcription factor and, accordingly, results in activation or repression of transcription .The effector-binding protein domains of transcriptional regulators appear to have evolved by distinct selective forces. In some cases, the effector-binding protein domains appear to derive from catalytic proteins, which may or may not retain the active site residues in their binding pockets during evolution and thus could possibly behave as bifunctional proteins. There are also a few examples of transcriptional regulators that might have evolved from enzymes that have lost their catalytic activity. In eukaryotes, we could mention the Gal80 regulator involved in the catabolism of galactose in Saccharomyces cerevisiae and Kluyveromyces lactis, and TAFII150 protein in Drosophila melanogaster which controls transcription mediated by the RNA polymerase (RNAP)-II. In prokaryotes, the HutC regulator of

Pseudomonas putida, involved in histidine utilization, and the FarR regulator of Escherichia coli, which controls the expression of Krebs cycle genes and responds to fatty acids, contain effector-binding domains similar to chorimaste lyases. However, the evolutionary pathways giving rise to these regulators have not yet been reproduced in the laboratory.

Anaerobic pathways provide reliable model scenarios to study protein evolution since they may preserve relics of ancient events occurring on early anaerobic Earth. In an effort to understand the evolution of regulators, we have reconstructed the most probable evolutionary pathway followed by the BzdR regulator. The BzdR protein of the facultative anaerobe β-Proteobacterium Azoarcus sp. CIB is the prototype of a new subfamily of transcriptional regulators. This regulator controls the expression of genes involved in the aerobic or anaerobic degradation of benzoate.

The predicted domain organization of BzdR consists of an N-terminal region (NBzdR, residues 1–90)，omologous to the DNA binding domain of members of the helix-turn-helix (HTH)-XRE transcriptional regulator, connected through a linker sequence to a C-terminal region (CBzdR, residues 131–298), This C-terminal region shows 23% sequence identity with the E. coli shikimate kinase I (SKI) (aroK gene product). In addition CBzdR conserves the SKI P-loop-containing nucleoside triphosphate hydrolase fold, the Walker-A motif and the Gly present in the Walker

B-motif of purine nucleotide-binding proteins, and recognizes the inducer molecule benzoyl-CoA. In this work, we have experimentally reproduced the most likely evolutionary pathway followed by the BzdR protein. First, we show that NBzdR and CBzdR are real functional domains able to bind DNA and the effector molecule, respectively. We then constructed functionally active synthetic BzdR-like regulators by fusing the DNA-binding domain of BzdR to E. coli SKI protein as the

effector-binding domain. The observed functionality of these synthetic regulators provides strong support for a role of an ancestral SK enzyme in the evolutionary origin of the BzdR protein.

译文：

一种酶演变成转录调节因子时一个缺失链的识别

摘要：

通过酶与DNA结合结构域结合的转录调节因子的演变是已被广泛接纳的观点。然而，很少有实验方法直接证明这一假说。在此，报告了在转录调节酶中的进化可信通路的重建。该BzdR蛋白是原核转录调节亚族的原始型，它控制包括苯甲酸厌氧降解基因的表达。已研究表明，BzdR蛋白的组成是由一个N-末端D NA结合结构域和C-末端效应结合结构域通过一个连接体连结，这显示了与莽草酸激酶(SK)有相当大的同一性。通过BzdR蛋白的DNA结合结构域与大肠杆菌莽草酸激酶I(SKI)蛋白相融合会构建活性合成BzdR蛋白相似调节蛋白，这有利地证明了在BzdR蛋白的进化起源中涉及到祖先的莽草酸激酶的结构域。为了进化成如今BzdR蛋白中的一个调节结构域，祖先的莽草酸激酶酶活的丧失是必要的。这还证明了酶先于可能控制其表达的调节系统而出现这一观点。

简介:

通过小分子的作用直接结合于转录因子的转录调控普遍存在于所有生命体中。大量的转录调节因子包含一个能与效应结合区融合的DNA结合结构域。与效应器结合可导致构象的改变，从而影响转录因子的特性，因而导致转录的激活或抑制。转录调节因子的效应结合蛋白结构域因不同的选择压力而进化产生。在一些情况下，效应结合蛋白结构域起源于催化蛋白。进化过程中，这些催化蛋白可能、也可能不保留它们结合区的活性位点残基，因而也可能表现为双官能蛋白。也有几个关于转录调节因子可能由丧失催化活性的酶演化而来的例子。在真核生物中，会提到存在于酿酒酵母和乳酸克鲁维酵母的半乳糖分解代谢中的Gal80调节因子以及在果蝇中通过RNA聚合酶Ⅱ介导的控制转录的TAFⅡ150蛋白。在原核生物中，恶臭假单胞菌的HutC调节因子、参与组氨酸利用和大肠杆菌的FarR 调节因子，其可控制三羧酸循环相关基因的表达和响应脂肪酸，含有相似于裂解酶的效应结合结构域。然而，进化途径中导致这些调节因子的产生尚未经实验证实。

厌氧通路给研究进化提供了可靠的模型场景，因为它们可能保留发生在早期厌氧地球远古事件的遗迹。在努力了解调节因子的进化过程中，我们已经建立了演化成BzdR调节因子的最可能的进化通路。兼性厌氧菌的β-变形菌纲固氮弧菌属物种的BzdR蛋白是转录调节因子的一个新亚族的雏形。该调节因子控制包括苯甲酸的需氧或厌氧讲解基因的表达。

预测的BzdR蛋白结构域组成是由一个N-末端区域(BzdR,残基1-90)，同源于螺旋-转角-螺旋(HTH)-XRE转录调节因子，通过一条连接序列与C-末端区域(CBzdR,残基131-298)结合，这一C-末端区显示与大肠杆菌莽草酸激酶（SKI）(aroK的基因产物)有23%的序列相同。此外，CBzdR保护了莽草酸激酶P-环核苷三磷酸水解酶折叠。沃克-A模体和甘氨酸呈现在嘌呤核苷酸结合蛋白的沃克-B模体中，识别诱导物分子苯甲酰基辅酶A。在此项研究中，我们通过实验再

现了BzdR蛋白形成之前的最可能的进化通路。首先，表明了NBzdR和CBzdR 能够分别与绑定DNA和效应分子结合的真正的功能结构域。接着，通过将BzdR 的DNA结合域与作为效应结合域的大肠杆菌莽草酸激酶蛋白相融合，构建了功能的活性合成BzdR类似调节因子。观察出的这些合成调节因子的函数性为BzdR 蛋白的进化起源中祖先链激酶的作用提供了强有力的证据。