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An investigation of ring-cleaving dioxygenase PcpA homologs and spectroscopic studies of the active site of PcpA
Livia Danae Amorosi
May 7, 2019
Department or Program
Many pesticides, wood preservatives, and insecticides polluting the environment are intractable because they are highly halogenated aromatic molecules. However, select soil bacteria have developed catabolic pathways to degrade some of these compounds and could potentially be used in bioremediation. Within certain pathways is a ring-cleaving dioxygenase (RCDO) that activates and inserts dioxygen into the aromatic compound, thus cleaving the ring. One such enzyme, PcpA (a type I HQDO) is found in the bacteria Sphingobium chlorophenolicum and preferentially cleaves halogenated substrates. This is unique in that most RCDOs are inactivated by halogenated substrates. Attempting to determine what features of PcpA afford it this uncommon functionality took two approaches. The first was the study of other type I HQDOs, to compare substrate specificity. Unfortunately attempts to study putative type I HQDOs 1ZSW, MnpC, MhqO, “CnpA”, LinEb and “LinEc” as well as type II HQDO HqdAB were ultimately unsuccessful. However, type II HQDO PnpC1C2 shows different substrate specificity and ring cleavage regiospecificity than PcpA, which is promising for future studies. The second approach used spectroscopic techniques to study an isotopically labeled inhibitor bound to the active site metal center. The T1 relaxation times of paramagnetic NMR are a function of the distance between the nuclei and the unpaired electrons of the metal. Thus, T1 relaxation times could be used to model the active site with an inhibitor bound, which is important as the known crystal structure of PcpA is of the protein with a sulfate ion bound. Unfortunately, due to rapid exchange between bound and free forms of the inhibitor, this experiment was ultimately unsuccessful. Future spectroscopic studies of the active site will be attempted using EPR, which will not face the issue of chemical exchange.