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.
