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.
|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|
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|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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