Enzymes containing mononuclear non-heme iron sites catalyze a diverse array of reactions that are significant to medicine and to the environment. This proposal describes plans to study three representatives of the largest, but perhaps least well understood, grouping of these enzymes: The a-ketoglutarate (aKG)-dependent dioxygenase superfamily. TauD catalyzes the hydroxylation of taunne and other sulfonates as a first step in their metabolism. TfdA carries out similar chemistry during catabolism of the herbicide 2,4-dichiorophenoxyacetic acid (2,4-D). Phytanoyl-CoA hydroxylase, a new research direction for this laboratory, is required for utilization of C-3 branched fatty acids; deficiencies of this human enzyme lead to Refsum disease, rhizomelic chondrodysplasia punctata, and ZeHweger syndrome.
The specific aims i nclude: (1) Characterize the enzyme mechanism of TauD and TfdA by examining the properties of catalytic intermediates and analyzing the effects of site-directed mutagenesis. (2) Examine the biogenesis of the tyrosyl radical, hydroxy-tryptophan, and histidyl-trihydroxyphenylalanine found in TauD and identify the structures & synthesis of modifications present in TfdA. (3) Obtain high-resolution three-dimensional protein structures of TauD, TfdA, and their variants in their various states. (4) Explore the metallocenter properties of phytanoyl-CoA hydroxylase using the recombinant human enzyme and/or a. more tractable microbial model system. Of particular interest will be studies to test a new mechanism for this enzyme superfamily. Specifically, we propose that these enzymes possess catalytically essential tyrosine residues that are used to resonance stabilize Fe(IV)=O as Fe(III)-OH/tyrosine radical states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063584-04
Application #
6874849
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Basavappa, Ravi
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
4
Fiscal Year
2005
Total Cost
$254,150
Indirect Cost
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
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
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 (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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