Nonheme diiron enzymes are involved in the synthesis of many biologically and industrially relevant compounds, including the formation of methanol, the biosynthesis of antibiotics, and the creation of DNA building blocks from RNA. The catalytic cycles of these enzymes involve the binding and subsequent activation of O2 to carry out a wide variety of chemistry. Importantly, the mechanism of these enzymes is thought to depend on the formation of a common (?-1,2-peroxo)diferric intermediate. This structure is proposed to be stable, requiring activation to a more reactive intermediate for subsequent catalysis. Although this enzyme superfamily has been studied for many years, several key mechanistic details surrounding how enzymes activate the common peroxo intermediate to maintain such diverse reactivity are unknown. The identification of new diiron enzymes within distinct protein folds and diiron coordination environments has prompted further investigation into the role of both the protein and the diiron site on formation and control of peroxo reactivity In particular, the Que laboratory has identified a peroxo intermediate in human deoxyhypusine hydroxylase (hDOHH). hDOHH catalyzes the post-translational hydroxylation of the amino acid hypusine. This amino acid is found only in the eukaryotic translational initiation factor 5A (eIF5A and is required for cell proliferation, making hDOHH an appealing target for cancer and HIV treatments. The hDOHH peroxo intermediate (hDOHHperoxo) is stable for days, making it the longest-lived peroxo species identified to date. Furthemore, the diiron cluster is housed in a protein fold and coordination environment unique from all other nonheme diiron enzymes. This project proposes to investigate mechanistic and structural details that regulate hDOHHperoxo activation using a host of biochemical and spectroscopic techniques. Specifically, this proposal will investigate how substrate binding, pH, and the distinct diiron coordination environment contribute to the formation and activation of hDOHHperoxo. The rates of hDOHHperoxo formation as a function of substrate concentration, varied pH and deuteration will be measured using UV-visible spectroscopy. Individual mutagenesis of four active site Glu ligands (Glu57, Glu90, Glu208, Glu241) to Asp and Gln will be performed to assess the contribution of electrostatics and sterics on hDOHHperoxo stability. Electronic and geometric details of the active site structure as a function of the above modifications (substrate, pH, coordinating ligand) will be ascertained using resonance Raman, M?ssbauer, and XAS spectroscopies. Importantly, these studies will be carried out for the first time using a peroxo species competent in carrying out the native chemistry of the enzyme. Information obtained from these experiments will add to our knowledge of features that affect the reactivity of peroxo intermediates, particularly those found in new protein folds and distinct diiron coordination environments, furthering our understanding of how O-O bond activation is accomplished and may lead to a better understanding of how to target hDOHH for cancer and HIV therapeutics.
Nonheme diiron enzymes activate dioxygen in order to catalyze a multitude of fundamentally critical biological reactions;in particular, this proposal i focused on understanding the mechanism of O2 activation in the human enzyme deoxyhypusine hydroxylase (hDOHH). hDOHH is involved in the post-translational synthesis of the modified amino acid hypusine, shown to be critical for eukaryotic cell proliferation. Further characterization of a diiron site in a new and different protein fold and coordination environment gives us the exciting opportunity to understand how nature has evolved different ways to carry out O-O activation and may lead to a better understanding of how to target this enzyme for cancer and HIV treatment.
| Jasniewski, Andrew J; Engstrom, Lisa M; Vu, Van V et al. (2016) X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a. J Biol Inorg Chem 21:605-18 |
