RUI: The Sources of Substrate Specificity in Hydroquinone Dioxygenases

利用细菌去除环境中的污染物，如多氯联苯（PCBs）和农药五氯酚（生物修复），引起了人们的极大兴趣。细菌酶可以分解这些化合物的稳定的六碳环，将它们从环境中清除。然而，分解氯化化合物的酶是罕见的，而且很少被描述。PcpA酶就是具有这种能力的酶之一。先前的研究表明，它对氯化化合物具有特异性，但它如何能够区分氯化和非氯化的化合物尚不清楚。拟议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为其他具有新特性的细菌工程方法提供见解，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。人们对酶如何识别氯化化合物知之甚少。对苯二酚双加氧酶，如PcpA，为了解氯化化合物的特异性来源提供了理想的平台。PcpA结合氯或溴取代基的底物和抑制剂比氟或甲基取代基的底物和抑制剂更好。一种假设是，一种很少被研究的相互作用，金属-卤素二次键，是造成这种特异性的原因。这项拟议的研究使用蛋白质晶体学、光谱学和量子化学计算来确定PcpA如何特异性地识别含有氯或溴取代基的对苯二酚，并激活这些化合物进行氧化环切割。对其他对苯二酚双加氧酶缺乏对氯化和溴化化合物的特异性的研究补充了这些调查的结果。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。这些结果为设计一种将儿茶酚双加氧酶转化为对苯二酚双加氧酶的特定酶的不同环切割活性提供了初步的努力。合成铁(II)-对苯二酚配合物用于显示对苯二酚质子化状态如何影响结合模式和与作为氧化环切割前体的二氧的反应性的基本方面。

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.