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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613226
• 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.

加拿大已经确定了22,000多个受污染或疑似受污染的地点并将其归类。它们中的许多含有卤代烃，这些污染物对自然分解和补救工作具有很强的抵抗力。其中一些化合物甚至可以在北极熊的血液中找到。脱卤酶是一种打破碳-卤键的酶；它们正在接受测试，以修复这些受污染的部位。通过我们的工作，我们打算改进这些蛋白质作为更环保的补救手段的使用。 许多细菌都会产生这样的脱卤酶，但仅仅通过基因组测序很难识别它们，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。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。为了对一些最顽固的环境污染物的生物降解解决方案的未来开发提供初步见解，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。后者中的四个属于名为HAD的蛋白质家族，是其被确认为脱氟酶的首批成员。我们的研究旨在确定为什么这些酶是如此有效的催化剂。 从沼泽细菌沼泽红假单胞菌中分离到一种脱卤酶，该酶属于另一个蛋白质家族ABH，并用X射线结晶学对其进行了详细的原子结构表征。这种酶将氟乙酸酯转化为无害的乙醇酸盐化合物。氟乙酸酯是一种有毒化合物，存在于澳大利亚、巴西和非洲的牧草中，用作杀虫剂。我们能够直观地看到蛋白质如何与其底物相互作用，从而组装出一组催化反应的“快照”。我们还可以证明，人们可以在不破坏晶体的情况下，在晶体中运行完整的催化循环。我们已经合成了修饰底物，通过将它们暴露在短脉冲激光下，可以将它们重新转变为活性底物。当这是在晶体中完成的，X射线照射到这些晶体上时，人们可以观看正在进行的催化反应的“分子电影”，从而能够观察到几乎原子细节的化学变化。 通过构建细菌或植物蛋白的嵌合体，这些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/可切换构建体的用途，这些构建体可以为脱卤酶反应增加可逆成分。我们还将测试不能脱氟的脱氯酶晶体，以确定它们在晶格中适应整个催化循环的能力。通过比较这两种“分子电影”，我们希望找出使脱氟酶成为脱氟酶的结构特征。