How enzymes break carbon-fluorine bonds
酶如何打破碳氟键
基本信息
- 批准号:170109-2010
- 负责人:
- 金额:$ 6.07万
- 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:2014
- 资助国家:加拿大
- 起止时间:2014-01-01 至 2015-12-31
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
Dehalogenase enzymes break carbon-halogen bonds. Most remarkable are enzymes able to break the most stable bond in organic chemistry, the carbon-fluorine bond. Supported by an NSERC Strategic Grant and in collaboration with E. Edwards (U of T) and A.Yakunin (SGC), we screened microbial genomes for dehalogenases. We raised the number of confirmed dehalogenases from 2 to 20, 9 of them defluorinases and 4 representing the first defluorinase members of the L-2-haloacid dehalogenase protein family. We will determine the molecular interactions that form the basis of their catalytic power, contributing to biodegradation solutions for most recalcitrant environmental pollutants. We have used ITC to determine kinetic parameters of native and mutant enzymes. The fluoroacetate dehalogenase RPA1163 from R. palustris was characterized in detail. Its static high-resolution crystal structures (~ 1.2 Å; native and mutants) with various ligands provided a set of 'snapshots' of the catalytic intermediates. They also indicated transient binding sites as the substrate approaches the catalytic machinery and rotational movements upon transformation. Our static analyses showed that the crystals accommodate the complete catalytic cycle without compromising diffraction power. We have synthesized the "caged" 1-(2-nitrophenyl) ethyl-derivative of the substrate. Short UV-laser pulses can transform this "caged compound" into the substrate, initiating its binding and catalytic conversion. Applying time- resolved (TR)-Laue techniques, we will collect diffraction data as a function of time since initiation and convert them to a "movie" of the catalytic reaction, allowing observation in almost atomic detail. We collaborate with the world's foremost experts in laser-triggered TR-Laue-diffraction to accelerate our progress and secure timely access to their superb resources. Our system will also serve as a test case for developing monochromatic methods for TR crystallography. In addition, we plan to engineer a light-sensitive dehalogenase by fusing a light-trigger LUV domain to the defluorinase, which will allow us to preform the substrate complex, thereby shifting the reaction from a bimolecular to a unimolecular one, allowing for easier data interpretation.
脱卤酶破坏碳-卤键。最引人注目的是能够打破有机化学中最稳定的键--碳-氟键的酶。在NSERC战略拨款的支持下,我们与E.Edwards(U Of T)和A.Yakunin(SGC)合作,筛选了微生物基因组中的脱卤酶。我们将已证实的脱卤酶从2个增加到20个,其中9个是脱氟酶,4个代表L-2-卤酸脱卤酶蛋白家族的第一个成员。我们将确定构成其催化能力基础的分子相互作用,有助于为大多数顽固的环境污染物提供生物降解解决方案。我们用ITC测定了天然和突变酶的动力学参数。对沼泽假单胞菌产生的氟乙酸酯脱卤酶RPA1163进行了详细的研究。它的静态高分辨晶体结构(~1.2;天然和突变体)与各种配体一起提供了一组催化中间体的“快照”。它们还指示了底物接近催化机械时的瞬时结合位置和转化时的旋转运动。静态分析表明,这些晶体在不影响衍射率的情况下适应了完整的催化循环。我们合成了底物的“笼状”1-(2-硝基苯基)乙基衍生物。短的紫外光激光脉冲可以将这种“笼状化合物”转化为底物,启动其结合和催化转化。应用时间分辨(Tr)-LAUE技术,我们将收集自启动以来随时间变化的衍射数据,并将它们转换为催化反应的“电影”,从而能够观察到几乎原子的细节。我们与世界上最顶尖的激光触发Tr-Laue衍射专家合作,以加快我们的进度,并确保及时访问他们的优质资源。我们的系统还将作为一个测试案例,用于开发单色的TR结晶学方法。此外,我们计划通过将光触发的LUV结构域融合到脱氟酶来设计一种光敏脱卤酶,这将使我们能够预形成底物复合体,从而将反应从双分子转移到单分子,从而允许更容易的数据解释。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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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:
$ 6.07万 - 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:
$ 6.07万 - 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:
$ 6.07万 - 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:2019
- 资助金额:
$ 6.07万 - 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:
$ 6.07万 - 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:
$ 6.07万 - 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:
$ 6.07万 - 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:
$ 6.07万 - 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:
$ 6.07万 - 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:
$ 6.07万 - 项目类别:
Discovery Grants Program - Individual
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