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 #
5R01GM063584-11
Application #
8538998
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
2013-09-01
Budget End
2014-08-31
Support Year
11
Fiscal Year
2013
Total Cost
$360,011
Indirect Cost
$115,442
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
Silvestrov, Pavel; Müller, Tina A; Clark, Kristen N et al. (2014) Homology modeling, molecular dynamics, and site-directed mutagenesis study of AlkB human homolog 1 (ALKBH1). J Mol Graph Model 54:123-30
Muller, Tina A; Andrzejak, Megan M; Hausinger, Robert P (2013) A covalent protein-DNA 5'-product adduct is generated following AP lyase activity of human ALKBH1 (AlkB homologue 1). Biochem J 452:509-18
Casey, Thomas M; Grzyska, Piotr K; Hausinger, Robert P et al. (2013) Measuring the orientation of taurine in the active site of the non-heme Fe(II)/*-ketoglutarate-dependent taurine hydroxylase (TauD) using electron spin echo envelope modulation (ESEEM) spectroscopy. J Phys Chem B 117:10384-94
Muller, Tina A; Yu, Kefei; Hausinger, Robert P et al. (2013) ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination. PLoS One 8:e67403
Simmons, Jana M; Koslowsky, Donna J; Hausinger, Robert P (2012) Characterization of a Trypanosoma brucei Alkb homolog capable of repairing alkylated DNA. Exp Parasitol 131:92-100
Mulrooney, Scott B; Howard, Michael J; Hausinger, Robert P (2011) The Escherichia coli alkylation response protein AidB is a redox partner of flavodoxin and binds RNA and acyl carrier protein. Arch Biochem Biophys 513:81-6
Simmons, Jana M; Koslowsky, Donna J; Hausinger, Robert P (2010) Trypanosoma brucei brucei: thymine 7-hydroxylase-like proteins. Exp Parasitol 124:453-8
Grzyska, Piotr K; Appelman, Evan H; Hausinger, Robert P et al. (2010) Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV=HO species. Proc Natl Acad Sci U S A 107:3982-7
Muller, Tina A; Meek, Katheryn; Hausinger, Robert P (2010) Human AlkB homologue 1 (ABH1) exhibits DNA lyase activity at abasic sites. DNA Repair (Amst) 9:58-65
Grzyska, Piotr K; Hausinger, Robert P; Proshlyakov, Denis A (2010) Metal and substrate binding to an Fe(II) dioxygenase resolved by UV spectroscopy with global regression analysis. Anal Biochem 399:64-71

Showing the most recent 10 out of 29 publications