How enzymes break carbon-fluorine bonds

酶如何打破碳氟键

基本信息

  • 批准号:
    RGPIN-2015-04877
  • 负责人:
  • 金额:
    $ 2.77万
  • 依托单位:
  • 依托单位国家:
    加拿大
  • 项目类别:
    Discovery Grants Program - Individual
  • 财政年份:
    2019
  • 资助国家:
    加拿大
  • 起止时间:
    2019-01-01 至 2020-12-31
  • 项目状态:
    已结题

项目摘要

Canada has identified and classified over 22,000 contaminated or suspected contaminated sites. Many of them harbour halogenated hydrocarbons, contaminants that are highly resistant to natural breakdown as well as remediation efforts. Some of these compounds can even be found in the blood of polar bears. Dehalogenases are enzymes that break carbon-halogen bonds; they are being tested for restoring such contaminated sites. With our work, we intend to improve the use of these proteins as more environmentally friendly means of remediation. ******Many bacteria produce such dehalogenases but it is difficult to identify them simply by sequencing the genome because they look very similar to other proteins that are not able to perform the same catalysis. Of special interest are enzymes that can break the strongest bond in organic chemistry, the one between carbon and fluorine. To provide initial insight towards the future development of biodegradation solutions for some of the most recalcitrant environmental pollutants, our lab, in collaboration with our colleague E. Edwards, screened bacteria for dehalogenase activity. We increased the number of confirmed dehalogenases from 2 to 20, 9 of them defluorinases. Four of the latter belong to a protein family called HAD and are its first members identified as defluorinases. Our research aims to determine why these enzymes are such potent catalysts. ******A dehalogenase belonging to another protein family called ABH was isolated from the marsh bacterium Rhodopseudomonas palustris and characterized in atomic detail by X-ray crystallography. The enzyme converts fluoroacetate, a toxic compound found in grasses in Australia, Brazil, and Africa and used as a pesticide, to the harmless compound glycolate. We were able to visualize how the protein interacts with its substrate to assemble a set of `snapshots' of the catalytic reaction. We could also show that one can run the complete catalytic cycle in the crystals without breaking them. We have synthesized modified substrates that can be turned back into active substrates by exposing them to short pulses of laser light. When this is done in crystals and X-rays are shone on these one can watch the `molecular movie' of the ongoing catalytic reaction, allowing the observation of the chemical transformation in almost atomic detail. ******By constructing chimeras of bacterial or plant proteins that change their shape upon illumination with various dehalogenases, we will test their use as light-activated/switchable constructs that could add a reversible component to the dehalogenase reaction. We will also test crystals of dechlorinating enzymes incapable of defluorination for their ability to accommodate the full catalytic cycle in the crystal lattice. By comparing both resulting "molecular movies", we hope to pinpoint the structural features that make a defluorinase a defluorinase.**
加拿大已查明22,000多个受污染或疑似受污染的场址并进行了分类。其中许多含有卤代烃,这种污染物很难自然分解,也很难进行补救。其中一些化合物甚至可以在北极熊的血液中找到。脱卤酶是一种能破坏碳-卤键的酶,目前正在测试它们是否能修复这些受污染的场地。通过我们的工作,我们打算改善这些蛋白质的使用,作为更环保的补救手段。** 许多细菌产生这样的脱卤酶,但很难简单地通过基因组测序来识别它们,因为它们看起来与其他不能进行相同催化的蛋白质非常相似。特别令人感兴趣的是酶,它可以打破有机化学中最强的键,即碳和氟之间的键。为了提供对一些最难降解的环境污染物的生物降解解决方案的未来发展的初步见解,我们的实验室,与我们的同事E。Edwards,筛选了具有脱卤酶活性的细菌。我们将确认的脱卤酶的数量从2个增加到20个,其中9个是脱氟酶。后者中的四个属于称为HAD的蛋白质家族,并且是其被鉴定为脱氟酶的第一个成员。我们的研究旨在确定为什么这些酶是如此有效的催化剂。****** 从沼泽细菌沼泽红球藻中分离出一种属于另一个蛋白质家族ABH的脱卤酶,并通过X射线晶体学对其进行了原子细节表征。 这种酶将氟乙酸盐(一种在澳大利亚、巴西和非洲的草中发现的有毒化合物,用作杀虫剂)转化为无害的乙醇酸盐。我们能够可视化蛋白质如何与其底物相互作用,以组装一组催化反应的“快照”。我们还可以证明,人们可以在晶体中运行完整的催化循环而不破坏它们。我们已经合成了改性的基板,可以通过将它们暴露于短脉冲的激光而变回活性基板。当这是在晶体中完成的,X射线照射在这些晶体上,人们可以观看正在进行的催化反应的“分子电影”,从而可以观察到几乎原子细节的化学转变。** 通过构建细菌或植物蛋白质的嵌合体,这些蛋白质在用各种脱卤酶照射时会改变它们的形状,我们将测试它们作为光激活/可切换构建体的用途,这些构建体可以为脱卤酶反应添加可逆组分。我们还将测试不能脱氯的脱氯酶晶体在晶格中容纳完整催化循环的能力。通过比较这两种“分子电影”,我们希望能够确定使脱氟酶成为脱氟酶的结构特征。

项目成果

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Pai, Emil其他文献

Pai, Emil的其他文献

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{{ truncateString('Pai, Emil', 18)}}的其他基金

Time-resolved crystallography of enzyme-catalyzed reactions
酶催化反应的时间分辨晶体学
  • 批准号:
    RGPIN-2020-06867
  • 财政年份:
    2022
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
Time-resolved crystallography of enzyme-catalyzed reactions
酶催化反应的时间分辨晶体学
  • 批准号:
    RGPIN-2020-06867
  • 财政年份:
    2021
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
Time-resolved crystallography of enzyme-catalyzed reactions
酶催化反应的时间分辨晶体学
  • 批准号:
    RGPIN-2020-06867
  • 财政年份:
    2020
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    RGPIN-2015-04877
  • 财政年份:
    2018
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    RGPIN-2015-04877
  • 财政年份:
    2017
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    RGPIN-2015-04877
  • 财政年份:
    2016
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    RGPIN-2015-04877
  • 财政年份:
    2015
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    170109-2010
  • 财政年份:
    2014
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    170109-2010
  • 财政年份:
    2013
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual
How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
  • 批准号:
    170109-2010
  • 财政年份:
    2012
  • 资助金额:
    $ 2.77万
  • 项目类别:
    Discovery Grants Program - Individual

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SSB 如何调节 YoaA-chi 的 DNA 损伤修复功能
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