RUI: The Sources of Substrate Specificity in Hydroquinone Dioxygenases

RUI：氢醌双加氧酶底物特异性的来源

• 批准号: 1506458
• 负责人: Timothy Machonkin
• 金额: $ 38.54万
• 依托单位: Whitman College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1506458&HistoricalAwards=false
• 关键词: RUI; Sources; Substrate; Specificity; Hydroquinone

There is much interest in the use of bacteria to remove pollutants such as polychlorinated biphenyls (PCBs) and the pesticide pentachlorophenol from the environment (bioremediation). Bacterial enzymes can break down the stable six-carbon ring of such compounds, removing them from the environment. However, enzymes that break down chlorinated compounds are rare and have been little characterized. The enzyme PcpA is one that has this capability. Previous research has shown that it is specific for chlorinated compounds, but how it is capable of distinguishing the chlorinated versus the non-chlorinated versions of a compound is not known. The proposed research seeks to understand the origin of this unusual specificity. Results of these efforts may provide insights into methods of engineering other bacteria with novel properties, for use in bioremediation or other applications. This work engages 8-12 Whitman College undergraduate students in cutting-edge research involving various biochemical techniques, providing them with important research training opportunities, including presenting their work at conferences and coauthoring publications.Little is known about how enzymes recognize chlorinated compounds. Hydroquinone dioxygenases, such as PcpA, provide an ideal platform for understanding the sources of specificity for chlorinated compounds. PcpA binds substrates and inhibitors with chlorine or bromine substituents much better than those with fluorine or methyl substituents. One hypothesis is that a little-studied interaction, metal-halogen secondary bonding, is responsible for this specificity. The proposed research uses protein crystallography, spectroscopy and quantum chemical calculations, to determine how PcpA specifically recognizes hydroquinones with chlorine or bromine substituents and activates these compounds for oxidative ring cleavage. The results of these investigations are complemented by studies on other hydroquinone dioxygenases that lack the specificity towards chlorinated and brominated compounds. Kinetics, mutagenesis, and substrate binding titrations are used to determine differences in substrate specificity and what factors are responsible for these differences. The results inform preliminary efforts to engineer different ring-cleaving activities into a given enzyme transforming a catechol dioxygenase into a hydroquinone dioxygenase. Synthetic iron(II)-hydroquinone complexes are used to show the fundamental aspects of how hydroquinone protonation state affects the binding mode and reactivity with dioxygen as a precursor to oxidative ring cleavage.

人们对利用细菌去除环境中的多氯联苯和农药五氯苯酚等污染物（生物修复）很感兴趣。细菌酶可以分解这些化合物的稳定六碳环，将它们从环境中清除。然而，分解氯化化合物的酶很少见，也很少被描述。PcpA酶就是具有这种能力的酶。以前的研究表明，它是特定的氯化化合物，但它是如何能够区分氯化与非氯化版本的化合物是未知的。这项拟议中的研究旨在了解这种不寻常的特异性的起源。这些努力的结果可能会提供深入了解工程的方法，其他细菌与新的属性，用于生物修复或其他应用。这项工作吸引了8-12名惠特曼学院的本科生参与涉及各种生物化学技术的前沿研究，为他们提供了重要的研究培训机会，包括在会议上展示他们的工作和共同撰写出版物。对苯二酚双加氧酶，如PcpA，为了解氯代化合物的特异性来源提供了一个理想的平台。PcpA与氯或溴取代基的底物和抑制剂的结合比与氟或甲基取代基的底物和抑制剂的结合好得多。一种假说是，一个很少研究的相互作用，金属-卤素二级键，是负责这种特异性。拟议的研究使用蛋白质晶体学，光谱学和量子化学计算，以确定PcpA如何特异性识别具有氯或溴取代基的氢醌，并激活这些化合物进行氧化环切割。这些调查的结果得到了对其他对苯二酚双加氧酶的研究的补充，这些酶对氯化和溴化化合物缺乏特异性。动力学、诱变和底物结合滴定用于确定底物特异性的差异以及导致这些差异的因素。结果通知初步的努力，工程师不同的环裂解活动到一个给定的酶转化成氢醌双加氧酶的儿茶酚双加氧酶。合成铁（II）-氢醌络合物用于显示氢醌质子化状态如何影响的结合模式和反应性与分子氧作为氧化环裂解的前体的基本方面。