Exploring reactivity and component interactions in Toluene/o-Xylene Monooxygenase from pseudomonas sp. OX1
Author(s)Liang, Alexandria Deliz
Massachusetts Institute of Technology. Department of Chemistry.
Stephen J. Lippard.
MetadataShow full item record
Chapter 1. Component Interactions in Three- and Four-Component Bacterial Multicomponent Monooxygenases. Bacterial multicomponent monooxygenases (BMMs) catalyze oxidation of hydrocarbon substrates through activation of dioxygen. Each BMM utilizes a diiron active site housed within a catalytic hydroxylase protein. This diiron active site is responsible for activation of dioxygen and oxidation of hydrocarbons. Additional component proteins modify the structure of the hydroxylase regulating substrate access and pre-organizing the diiron site for reactivity. The relationships between structure and reactivity that provide this control are reviewed for both three- and four-component BMMs, including soluble methane monooxygenase, phenol hydroxylase, toluene 4-monooxygenase, and toluene/oxylene monooxygenase. Comparisons between three- and four-component BMMs are highlighted to demonstrate how nature preserves the control over reactivity within the BMM superfamily. Chapter 2. A Flexible Glutamine Regulates the Catalytic Activity of Toluene/o- Xylene Monooxygenase. Toluene/o-xylene monooxygenase (ToMO) belongs to the enzyme superfamily of bacterial multicomponent monooxygenases (BMMs) and is capable of oxidizing aromatic substrates. The carboxylate-rich diiron active site is located 12 A below the surface of the catalytic hydroxylase component (ToMOH). The shortest opening between the surface of the protein and the diiron active site is a small hydrophilic pore. Here we examine the function of residues lining this pore, N202 and Q228, within ToMOH from Pseudomonas sp. OX1. Through characterization of the steady-state turnover of WT ToMOH and three mutant enzymes, N202A, Q228A, and Q228E, we demonstrate that these residues are critical for turnover. Mechanistic analysis reveals that these residues are critical for water egress and efficiently consuming NADH to hydroxylate product. We propose that this activity results from movement of these residues, opening and closing the pore during catalysis. In addition, N202 and Q228 are important for interaction of two component proteins, the diiron-reducing protein and the regulatory protein, suggesting that these two proteins bind competitively to the hydroxylase. The function of the pore region in other BMMs is discussed in light of these results. Chapter 3. Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase. Toluene/o-xylene monooxygenase (ToMO) activates dioxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, the diiron active site must acquire two electrons. This process requires three redox active proteins, a hydroxylase (ToMOH), a Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). A fourth, regulatory component with no redox active cofactors is also required to achieve catalysis (ToMOD). Through pre-steady-state kinetics, we demonstrate that ToMOD alters electron transfer from ToMOC to ToMOH. Under steady-state conditions, ToMOD increases the rate of turnover up to one equivalent of ToMOD to ToMOH. At excess ToMOD concentrations, the regulatory protein inhibits steady-state catalysis in a manner that depends upon the concentration of ToMOC. Protein-binding studies, computational docking, and rapid electron transfer kinetics indicate that this inhibitory function results from competition between ToMOD and ToMOC for binding to ToMOH. These results are discussed in the context of additional proteins in the bacterial multicomponent monooxygenase superfamily. Chapter 4. Oxygen Activation by the Hydroxylase of Toluene/o-Xylene Monooxygenase in the Presence of its Redox Partners. To hydroxylate arene substrates, toluene/o-xylene monooxygenase (ToMO) utilizes four protein components, a catalytic hydroxylase (ToMOH), a regulatory protein (ToMOD), a Rieske protein (ToMOC), and a reductase (ToMOF). Within ToMOH, this chemistry is achieved through the activation of dioxygen. Previous dioxygen activation studies of ToMO have utilized a simplified protein system comprising ToMOH and ToMOD, but with dithionite and methyl viologen supplying the electrons. Here, we revisit the dioxygen activation experiments but with ToMOC, ToMOF, and NADH. The use of these proteins and NADH dramatically alters dioxygen activation chemistry and subsequent arene hydroxylation in single turnover studies. Chapter 5. Oxygen Activation in the T201S Variant of Toluene/o-Xylene Monooxygenase. The secondary coordination spheres in diiron proteins influence reactivity at the active site. In the diiron protein toluene/o-xylene monooxygenase, a threonine residue (T201) near the diiron site modulates steady-state turnover and dioxygen activation chemistry in the presence and absence of substrate. Previous oxygen-activation studies, reveal that mutation of this residue to a serine (T201S) yields diiron-0 2 adducts different from those observed for of WT ToMOH. These oxygen-activation experiments were conducted using dithionite and methyl viologen as the reducing agents. As in Chapter 4, we re-examine oxygen activation by T201S in the presence of the redox proteins, ToMOC and ToMOF. Stopped-flow UVvisible spectroscopy reveals that the use of these component proteins changes the number and identity of diiron-02 adducts formed during the dioxygen activation steps in T201S. Appendix A. Heterologous Expression and Purification of Components of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. OXI. Here we provide the detailed protocols for expression and purification of the component proteins of toluene/o-xylene monooxygenase. Gene sequences and miscellaneous purification and handling notes are also provided.
Thesis: Ph. D. in Inorganic Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Department of Chemistry
Massachusetts Institute of Technology