RUI: The Sources of Substrate Specificity in Hydroquinone Dioxygenases

利用细菌从环境中去除多氯联苯和杀虫剂五氯苯酚等污染物引起了人们的极大兴趣(生物修复)。细菌酶可以分解这类化合物的稳定的六碳环，将它们从环境中移除。然而，分解氯化化合物的酶很少见，也很少被描述。PCPA就是一种具有这种能力的酶。以前的研究表明，它是氯化化合物特有的，但它如何能够区分化合物的氯化和非氯化版本尚不清楚。这项拟议的研究试图理解这种不同寻常的特殊性的起源。这些努力的结果可能为设计其他具有新特性的细菌的方法提供洞察力，用于生物修复或其他应用。这项工作吸引了8-12名惠特曼学院本科生从事涉及各种生化技术的尖端研究，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，为了解氯代化合物的专一性来源提供了一个理想的平台。PCPA与氯或溴取代基结合底物和缓蚀剂的效果比与氟或甲基取代基结合的好得多。一种假设是，这种特异性是由一种鲜为人知的相互作用，即金属-卤素二次键造成的。这项拟议的研究使用蛋白质结晶学、光谱学和量子化学计算来确定PCPA如何专门识别具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断裂。这些研究的结果得到了其他氢醌双加氧酶的研究的补充，这些研究缺乏对氯化和溴化化合物的特异性。使用动力学、诱变和底物结合滴定来确定底物专一性的差异以及导致这些差异的因素。这些结果提供了初步的努力，将不同的裂环活性工程到给定的酶中，将邻苯二酚双加氧酶转化为对苯二酚双加氧酶。合成的铁(II)-对苯二酚络合物被用来显示对苯二酚质子化状态如何影响作为氧化环裂解前体的氧的结合方式和反应活性的基本方面。

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.