Metal Ion Binding and Substrate Specificity in Hydroquinone Dioxygenases
EMSL Project ID
36402
Abstract
Bacterial pathways for the catabolism of aromatic hydrocarbons are of interest for use in bioremediation of pollutants such as chlorinated arenes. The oxidative ring cleavage step is often the major bottleneck. One such class of oxidative ring-cleaving enzymes are the hydroquinones dioxygenases (HQDOs). These enzymes have been little studied, and the factors that determine their unique substrate specificity remain unknown. The HQDOs are of particular interest for three reasons. (1) The nature of the substrate precludes bidentate coordination, indicating that there must be significant differences in substrate binding and cleavage compared to the well-studied catechol extradiol dioxygenases (EDOs). (2) While in the EDOs, chlorinated catechols usually lead to mechanism-based inactivation and thus are not substrates, in the HQDOs, chlorinated rings are the preferred (and in some cases, the only) substrates. (3) Since these enzymes are involved in the catabolism of xenobiotic compounds such as pentachlorophenol, this enzymatic function presumably evolved recently from some other enzyme class. We propose to use paramagnetic NMR as a direct spectroscopic probe for measuring Fe(II) and substrate/inhibitor binding to the active site of two hydroquione dioxygenases, PcpA and LinE, as well as a structurally characterized homolog that lacks ring-cleaving ability. We will measure Fe(II)-binding by monitoring the appearance of hyperfine-shifted peaks in a 1D 1H-NMR experiment. Similar paramagnetic 1D 1H-NMR will be used to measure the binding affinity of substrates and inhibitors to the Fe(II) center in these enzymes. Additionally, we will label the substrate with 2H and acquire 2H-NMR spectra. One issue of great interest is if monosubstituted hydroquinones bind in two different orientations, since this may partly explain the difference in substrate specificity between PcpA and LinE. Detailed analyses of the hyperfine shifts may allow assignment of the peaks to specific ring orientations. Understanding of how PcpA and LinE are able to specifically recognize and oxidatively cleave chlorinated aromatic rings could lead to important insights that would enable the redesign of other ring-cleaving dioxygenases with improved activity on chlorinated rings, which in turn could lead to improved bacterial bioremediation pathways for chlorinated organic pollutants. Access to the NMR spectrometers at EMSL (mostly the 600 MHz spectrometer, but possibly the 750 or 800 MHz instrument as well) is necessary for this work, since an NMR spectrometer is not currently available at Whitman College.
Project Details
Project type
Exploratory Research
Start Date
2009-10-07
End Date
2010-10-10
Status
Closed
Released Data Link
Team
Principal Investigator