This project examines the enzymatic mechanism used by FeII/?-ketoglutarate (?KG)-dependent hydroxylases and explores the diversity of reactions they catalyze. Members of this enzyme family are widespread in bacteria and eukaryotes (including humans) where they promote reactions of fundamental importance including DNA/RNA repair, synthesis/degradation of a vast repertoire of small molecules, lipid metabolism, and protein hydroxylation related to oxygen sensing, chromatin demethylation, or structural interactions. The studies detailed in this proposal focus on four aims. First, we will define the chemical steps during early catalysis by applying an innovative continuous- flow Raman spectroscopic approach to TauD, the best studied member of this enzyme family. Of special interest will be the properties of a key TauD variant that slowly forms the known FeIV=O intermediate, as well as the behavior of a thermophilic homologue. Parallel studies will probe for uniformity of the identified reaction intermediates in two other available family members. Second, pulsed EPR techniques will be utilized to investigate the geometries of active site environments for enzymes with bound nitric oxide (NO), a surrogate of O2. Measurements using these novel methods will be validated with TauD, where we have crystallographic information, and then applied to XanA, a xanthine-degrading enzyme, for which structural data are lacking. In particular, these techniques will be exploited to probe small structural changes at the active site upon substrate binding or in selected variant proteins. Third, the presence of a second FeII binding site in TauD will be confirmed and the function of this site will be investigated. As part of these studies, we will explore the use of phosphorescence quenching to obtain thermodynamic binding data on anaerobic proteins. Finally, biochemical and spectroscopic properties will be elucidated for TET1, a 5-meC hydroxylase that might function with another enzyme as a DNA demethylase. In total, this work will enhance our understanding of the enzyme mechanism common to this versatile enzyme family while further defining new and diverse roles for its individual members. Such studies have medical relevance because understanding of this mechanism is critical for developing treatments of human genetic diseases associated with defects in FeII/?KG hydroxylases, for defending against pathogens where such enzymes play essential roles, and for optimizing the synthesis of antibiotics by these enzymes in other microbes.

Public Health Relevance

FeII/?-ketoglutarate dependent hydroxylases catalyze dozens of reactions in humans, and defective enzymes underlie several human genetic diseases. Many pathogens of humans contain key representatives of this enzyme family, and an improved understanding of their catalytic mechanisms will facilitate the development of new drugs for controlling infections. Other microbes use these enzymes to synthesize antibiotics, offering the potential to engineer these catalysts for creating novel antimicrobial agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063584-10
Application #
8338809
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Smith, Ward
Project Start
2001-07-01
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
10
Fiscal Year
2012
Total Cost
$373,071
Indirect Cost
$119,632
Name
Michigan State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
Herr, Caitlyn Q; Hausinger, Robert P (2018) Amazing Diversity in Biochemical Roles of Fe(II)/2-Oxoglutarate Oxygenases. Trends Biochem Sci 43:517-532
Müller, Tina A; Struble, Sarah L; Meek, Katheryn et al. (2018) Characterization of human AlkB homolog 1 produced in mammalian cells and demonstration of mitochondrial dysfunction in ALKBH1-deficient cells. Biochem Biophys Res Commun 495:98-103
Henderson, Kate L; Li, Mingjie; Martinez, Salette et al. (2017) Global stability of an ?-ketoglutarate-dependent dioxygenase (TauD) and its related complexes. Biochim Biophys Acta Gen Subj 1861:987-994
Martinez, Salette; Fellner, Matthias; Herr, Caitlyn Q et al. (2017) Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist. J Am Chem Soc 139:11980-11988
Martinez, Salette; Hausinger, Robert P (2017) Correction to Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2. Biochemistry 56:3158
Müller, Tina A; Tobar, Michael A; Perian, Madison N et al. (2017) Biochemical Characterization of AP Lyase and m6A Demethylase Activities of Human AlkB Homologue 1 (ALKBH1). Biochemistry 56:1899-1910
Walker, Alice R; Silvestrov, Pavel; Müller, Tina A et al. (2017) ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding. PLoS Comput Biol 13:e1005345
Proshlyakov, Denis A; McCracken, John; Hausinger, Robert P (2017) Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study. J Biol Inorg Chem 22:367-379
Martinez, Salette; Hausinger, Robert P (2016) Biochemical and Spectroscopic Characterization of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme from Pseudomonas syringae pv. phaseolicola PK2. Biochemistry 55:5989-5999
Henderson, Kate L; Müller, Tina A; Hausinger, Robert P et al. (2015) Calorimetric assessment of Fe(2+) binding to ?-ketoglutarate/taurine dioxygenase: ironing out the energetics of metal coordination by the 2-His-1-carboxylate facial triad. Inorg Chem 54:2278-83

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