Graduation Semester and Year




Document Type


Degree Name

Doctor of Philosophy in Chemistry


Chemistry and Biochemistry

First Advisor

Brad S Pierce


Considerable research interest has focused on the non-heme oxygenase/oxidase enzyme family because of vast array of chemically diverse O2-depedent oxidations they participate in. For example, the bacterial multicomponent monooxygenases (sMMOH and ToMOH) are bacterial diiron enzymes that are involved in the oxidations of simple carbon sources (methane and toluene) for nutrition and other cellular cycles. Investigations of the BMM systems have significant implications for the research and development in greenhouse emissions. The hydroxylase component of the BMM superfamily is coordinated by 2-Histidine-4-carboxylate ligands (Asp or Glu) [2H4C]. The [2H4C] configuration is the typical moiety for the 2-electron oxidation in the monooxygenases. Following reduction, the diferrous cluster is able to catalyze the O2-depedent oxidation of the respective substrate by 2-electrons. Much like the bacterial multicomponent monooxygenase (BMM) superfamily, the binuclear site of Salmonella typhimurium MiaE is coordinated by 2-His-4-carboxylate (Asp or Glu) residues (2H4C). The Pierce group has extensively characterized the wild-type MiaE and its native, 2-electron tRNA-hydroxlase activity with a variety of spectroscopic and analytical methods (EPR, 57Fe Mӧssbauer, Circular dichroism (CD), UV-vis, X-ray diffraction (XRD), HPLC/LCMS, steady-state kinetics). With recent discoveries of the arylamine N-oxygenases, AurF and CmlI, the interests shifted toward the potential functionality of MiaE as a model for arylamine N-oxygenase chemistry. Comparisons of crystal structures with AurF suggests that the MiaE L199H variant can re-create the 3-His-4-carboxylate (3H4C) environment in the binuclear site. DFT studies on the wild-type MiaE revealed the shift in the F181 side chain for incorporation of the histidine residue and does not significantly affect the geometry of the active-site. Spectroscopic studies (UV-vis, EPR, and Mӧssbauer) of the L199H variant show good agreement with spectroscopic values reported for other arylamine N-oxygenases. Reactivity studies showed successful incorporation of molecular oxygen into an arylamine substrate with expected mass shift through HPLC and LC-MS. Together, this research has shown the potential for MiaE to serve as the framework for further investigation into arylamine N-oxygenase chemistry biologically inspired designed for catalyst development. Our research group has had significant interests in the role of thiol dioxygenases (TDOs) in the role of sulfur regulation. Clinical studies have shown a correlation between sulfur imbalances and the development of neurodegenerative diseases. TDOs are involved in the first irreversible step in the oxidation of sulfur-containing amino acid derivatives. The mammalian enzyme, cysteamine dioxygenase (ADO), is a non-heme iron enzyme that catalyzes the O2-depedent oxidation of cysteamine to produce hypotaurine. Hypotaurine and taurine biosynthesis are important metabolites for brain and cellular development. Extenstive studies have been dedicated to the mammalian counterpart cysteine dioxygenase (CDO). Consequently, ADO remains a relatively uncharacterized member of the non-heme iron family. Sequence homology between CDO and ADO show a conserved first-coordination sphere suggesting comparable mechanistic features for catalysis and substrate binding. Steady-state and NMR studies on ADO shows a proclivity for the native substrate, cysteamine (ca) with little reactivity toward L-cysteine (cys). Additionally, key ionization events in kcat and kcat/KM suggests a different collection of residues involved in catalysis that differs from what is seen in other thiol dioxygenases. The complementary X-band EPR studies suggests the substrate does not bind directly to the Fe-site. Together, these studies suggest potential deviations in mechanistic steps during catalysis from what is classically observed in thiol dioxygenases.


sMMOH, soluble methane monooxygenase, ToMOH, toluene monooxygenases


Chemistry | Physical Sciences and Mathematics


Degree granted by The University of Texas at Arlington

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