结核分枝杆菌螺旋酶GyrA与GyrB互作区域的缺失突变体对酶功能的影响
发布时间：2018-11-25 12:06
【摘要】：DNA螺旋酶是唯一一种可以引入DNA负超螺旋的type II拓扑异构酶,它是原核细胞所必须的基本酶类,参与了细胞内DNA的生理活动,如DNA复制、转录、基因重组、染色体重组等生理活动,因人体不存在DNA螺旋酶,螺旋酶被开发作为很多抗癌药物、抗生素类药物、多肽类蛋白的作用靶标,如喹喏T美嘁┪铩被愣顾乩嘁┪铩fpA Mt和QnrB4等。但是由于标准抗结核药物已持续使用了几十年,使得结核分枝杆菌这些药物的耐药性越来越大,产生了耐多药结核病和广泛耐药结核病,给结核病的治疗带来了很大的困难,人们需要开发更有效、更有针对性的药物。 DNA螺旋酶是由GyrA和GyrB两个亚基组成的四聚体,单独的GyrA或GyrB亚基不发挥其酶活特性,只有重组后才具有酶活功效。为了针对结核病的治疗开发有效的药物,我们需要药物作用靶标——DNA螺旋酶更有具体的信息。目前,对螺旋酶的GyrA和GyrB亚基的相互作用区域、结构、位点等的研究并不是很深入,为了研究GyrB与GyrA亚基的相互作用位点,对构建的一系列GyrB亚基的突变体进行了全酶酶活性的测定,以确定GyrB和GyrA亚基的相互作用区域。本课题研究结果如下： 1、野生型GyrA亚基、GyrB亚基、GyrB亚基一系列突变体(GyrB1-518、 GyrB1-531、GyrB△531-550、GyrB△548-564、GyrB-CTD、GyrB-NTD)的诱导表达、纯化、浓缩、浓度测定。 2、确定本实验室条件下测定DNA螺旋酶松弛活性和切割活性的反应条件和反应体系的主要成分,松弛活性为37℃、30min,切割活性为37℃、20min,反应体系中亚精胺、DTT、tRNA为必须成分,EDTA可以省略。 3、GyrB亚基C端是其与GyrA的相互作用的关键区域,531-550和548-564位氨基酸在松弛活性和切割活性中的重要作用,它们均位于GyrB亚基Toprim结构域中,Toprim结构域是GyrA和GyrB相互作用的关键结构域。对其中的位点进行缺失突变后发现,531-550区域含有DxD的功能模式位点,它的作用同时表现在对酶活的影响和结构的支撑上；而548-564位氨基酸的功能更多的是表现在对结构的支撑和构造上。
[Abstract]:DNA helicase is the only type II topoisomerase that can introduce DNA negative superhelix. It is the basic enzyme necessary for prokaryotic cells and participates in the physiological activities of intracellular DNA, such as DNA replication, transcription, gene recombination. Chromosome recombination and other physiological activities, because the human body does not exist DNA helicase, helicase has been developed as a variety of anticancer drugs, antibiotics, polypeptide protein targets, such as quine T scalloping? By? FpA Mt, QnrB4, etc. However, because the standard anti-tuberculosis drugs have been used for decades, the drug resistance of Mycobacterium tuberculosis has become more and more serious, resulting in multi-drug-resistant tuberculosis and widely drug-resistant tuberculosis, which has brought great difficulties to the treatment of tuberculosis. People need to develop more effective and targeted drugs. DNA helicase is a tetramer composed of two subunits of GyrA and GyrB. The single GyrA or GyrB subunit does not play its enzyme activity characteristic, only after recombination has the enzyme activity function. In order to develop effective drugs for the treatment of tuberculosis, we need more specific information about the target of drug action-DNA helicase. In order to study the interaction sites between GyrB and GyrA subunits, the interaction regions, structures and loci of GyrA and GyrB subunits of helicases are not well studied. The total enzyme activity of a series of GyrB subunit mutants was determined to determine the interaction region between GyrB and GyrA subunits. The results are as follows: 1. A series of mutants (GyrB1-518, GyrB1-531,GyrB 531-550 GyrB 548-564GyrB-CTDGyrB-NTD) of wild type GyrA subunits, GyrB subunits and GyrB subunits were induced, purified and concentrated. Concentration determination. 2. The reaction conditions and main components of DNA helicase relaxation activity and cleavage activity were determined in our laboratory. The relaxation activity was 37 鈩，
本文编号：2356041
[Abstract]:DNA helicase is the only type II topoisomerase that can introduce DNA negative superhelix. It is the basic enzyme necessary for prokaryotic cells and participates in the physiological activities of intracellular DNA, such as DNA replication, transcription, gene recombination. Chromosome recombination and other physiological activities, because the human body does not exist DNA helicase, helicase has been developed as a variety of anticancer drugs, antibiotics, polypeptide protein targets, such as quine T scalloping? By? FpA Mt, QnrB4, etc. However, because the standard anti-tuberculosis drugs have been used for decades, the drug resistance of Mycobacterium tuberculosis has become more and more serious, resulting in multi-drug-resistant tuberculosis and widely drug-resistant tuberculosis, which has brought great difficulties to the treatment of tuberculosis. People need to develop more effective and targeted drugs. DNA helicase is a tetramer composed of two subunits of GyrA and GyrB. The single GyrA or GyrB subunit does not play its enzyme activity characteristic, only after recombination has the enzyme activity function. In order to develop effective drugs for the treatment of tuberculosis, we need more specific information about the target of drug action-DNA helicase. In order to study the interaction sites between GyrB and GyrA subunits, the interaction regions, structures and loci of GyrA and GyrB subunits of helicases are not well studied. The total enzyme activity of a series of GyrB subunit mutants was determined to determine the interaction region between GyrB and GyrA subunits. The results are as follows: 1. A series of mutants (GyrB1-518, GyrB1-531,GyrB 531-550 GyrB 548-564GyrB-CTDGyrB-NTD) of wild type GyrA subunits, GyrB subunits and GyrB subunits were induced, purified and concentrated. Concentration determination. 2. The reaction conditions and main components of DNA helicase relaxation activity and cleavage activity were determined in our laboratory. The relaxation activity was 37 鈩，
本文编号：2356041
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