How enzymes break carbon-fluorine bonds

酶如何打破碳氟键

• 批准号: RGPIN-2015-04877
• 负责人: Pai, Emil
• 金额: $ 2.77万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=661406
• 关键词: How; enzymes; break; carbon; fluorine

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.**

加拿大已经确定并分类了超过22,000个受污染或疑似受污染的地点。其中许多都含有卤化碳氢化合物，这种污染物对自然分解和补救措施都具有很强的抵抗力。其中一些化合物甚至可以在北极熊的血液中找到。脱卤酶是破坏碳-卤素键的酶；他们正在接受测试，以恢复这些受污染的场所。通过我们的工作，我们打算改善这些蛋白质的使用，作为更环保的修复手段。******许多细菌产生这样的去卤酶，但很难通过简单的基因组测序来识别它们，因为它们看起来与其他无法执行相同催化作用的蛋白质非常相似。人们特别感兴趣的是那些能破坏有机化学中最强键的酶，即碳和氟之间的键。为了对一些最顽固的环境污染物的生物降解解决方案的未来发展提供初步的见解，我们的实验室与我们的同事E. Edwards合作，筛选了细菌的脱卤酶活性。我们将确认的去卤酶的数量从2个增加到20个，其中9个是去氟酶。后者中的四种属于HAD蛋白家族，是该家族的第一批成员，被确定为去氟酶。我们的研究旨在确定为什么这些酶是如此有效的催化剂。******从沼泽细菌红假单胞菌（Rhodopseudomonas palustris）中分离出一种名为ABH的蛋白质家族的脱卤酶，并通过x射线晶体学对其进行了原子细节表征。氟乙酸是一种有毒的化合物，存在于澳大利亚、巴西和非洲的草中，被用作杀虫剂。这种酶将氟乙酸转化为无害的化合物乙醇酸。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，人们可以在不破坏晶体的情况下，在晶体中进行完整的催化循环。我们已经合成了经过修饰的衬底，通过将它们暴露在短脉冲激光下，它们可以变回有活性的衬底。当这在晶体中完成，x射线照射在这些晶体上时，人们可以看到正在进行的催化反应的“分子电影”，允许在几乎原子的细节上观察化学转化。******通过构建细菌或植物蛋白嵌合体，在各种脱卤酶的照射下改变其形状，我们将测试它们作为光激活/可切换结构的用途，这些结构可以为脱卤酶反应添加可逆成分。我们还将测试不能除氟的脱氯酶晶体，因为它们能够在晶格中容纳完整的催化循环。通过比较两种结果的“分子电影”，我们希望能精确地指出使去氟酶成为去氟酶的结构特征