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首页> 外文学位 >High-throughput rational protein engineering generates organophosphorus hydrolase variants with improved activity.
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High-throughput rational protein engineering generates organophosphorus hydrolase variants with improved activity.

机译：高通量的理性蛋白质工程产生具有改善的活性的有机磷水解酶变体。

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摘要

Organophosphate compounds belong to a lethal class of chemicals that have been employed as agricultural pesticides and chemical warfare agents. As they are a major threat to human health, there is an urgent need for enzymes that will efficiently degrade and detoxify particular organophosphate compounds. A naturally occurring enzyme, organophosphorus hydrolase (OPH), has been shown to hydrolyze paraoxon, an organophosphorus pesticide, at a nearly diffusion-limited rate. OPH displays weak activity against other pesticides, such as methyl parathion and coumaphos, as well as nerve agents, such as soman, sarin, and Russian VX. Thus, OPH is an attractive target for site-directed mutagenesis aimed at achieving sufficient levels of activity for bioremediation and detoxification.; In this dissertation, a universal gene engineering system, terminator overhang PCR cloning (TOPC), is used to construct a customized OPH expression plasmid and generate a library of OPH variants. With the addition of automated protein expression and assay, TOPC represents a high-throughput, iterative process of design, production, and testing. Knowledge gained during each iteration is used to modify design parameters for the next iteration. In the initial iteration of the OPH project, described here, 86 single amino acid substitutions were engineered into the OPH gene. The resulting variants were tested for their ability to cleave the preferred OPH substrate, paraoxon, and two inefficiently cleaved substrates, methyl parathion and coumaphos. A single iteration of the process was sufficient to produce three variants that cleave methyl parathion with markedly increased efficiency. In contrast, a variant that efficiently cleaves coumaphos was not identified. One variant, H254R, cleaved methyl parathion with an initial velocity 12-fold greater than the natural enzyme. Kinetic analysis of this variant revealed that substrate specificity (Kcat/K m) was increased 33-fold, mainly attributable to a 24-fold increase in Kcat. One round of protein engineering was sufficient to increase substrate specificity against methyl parathion to within 13% of the substrate specificity demonstrated by the wild type enzyme against paraoxon. Additional rounds of large-scale protein mutagenesis using the TOPC strategy should allow for further productive rational engineering of OPH.
机译：有机磷酸酯化合物属于致命类化学物质，已被用作农业杀虫剂和化学战剂。由于它们是对人类健康的主要威胁，因此迫切需要能够有效降解和解毒特定有机磷酸酯化合物的酶。天然存在的酶有机磷水解酶（OPH）已显示出以几乎扩散受限的速率水解有机磷农药对氧磷。 OPH对其他农药（如甲基对硫磷和香豆磷）以及神经毒剂（如梭曼，沙林和俄罗斯VX）的活性较弱。因此，OPH是定点诱变的诱人靶标，旨在实现足够水平的生物修复和解毒活性。本文采用通用的基因工程系统，即终止子突出PCR克隆（TOPC），构建了定制的OPH表达质粒，并构建了OPH变体文库。加上自动化的蛋白质表达和测定，TOPC代表了设计，生产和测试的高通量迭代过程。在每次迭代中获得的知识可用于修改下一个迭代的设计参数。在这里描述的OPH项目的初始迭代中，将86个单氨基酸替换工程化到OPH基因中。测试了所得变体裂解优选的OPH底物对氧磷和两种裂解效率低下的底物甲基对硫磷和库玛磷的能力。该过程的单次迭代足以产生三个裂解甲基对硫磷的变体，效率显着提高。相反，未鉴定出有效裂解香豆磷的变体。一种变体H254R裂解了甲基对硫磷，其初始速度是天然酶的12倍。此变体的动力学分析表明，底物特异性（Kcat / K m）增加了33倍，主要归因于Kcat增加了24倍。一轮蛋白质工程足以将针对甲基对硫磷的底物特异性提高至野生型针对对氧磷酶所证明的底物特异性的13％之内。使用TOPC策略进行的额外几轮大规模蛋白质诱变，应可进一步进行OPH的生产性合理工程。

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  • 作者

    Turczyk, Brian Michael.;

  • 作者单位

    Boston University.;

  • 授予单位 Boston University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 145 p.
  • 总页数 145
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;
  • 关键词

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