This project is a multi-disciplinary study of the structure and function of oxygenases and other redox enzymes. The principle focus is on how oxygen is activated by Rieske oxygenases and on cytochromes P450. In addition, collaborative studies of other metalloproteins are also being carried out. Oxygenases are found in all aerobic organisms and are important in the biosynthesis, transformation, and degradation of steroids, nucleic acids, catecholamines, collagen, drugs, prostaglandins, lignin, and various foreign compounds. Thus, these enzymes are crucial to a majority of aerobic life forms and are requisite to the development of bioremediation processes necessary for dealing with pollution in our environment, one of the major health problems of the world.We will investigate in detail the physical, chemical, and kinetic properties of phthalate dioxygenase (PDO), a paradigm for the Rieske oxygenases that catalyze the first step in the aerobic metabolism of many aromatic compounds. In addition to their role in biodegradation, the products of Rieske nonheme iron-containing enzyme catalysis are often cis-dihydrodiols, which are valuable in """"""""green"""""""" synthetic chemistry. We will characterize intermediates that are involved in the oxygenation reaction. In addition, we will investigate (in collaboration with M.J. Coon and J. Dawson) several aspects of intermediates involved in oxygenation processes by cytochromes P450. Our hypothesis is that parallel studies of these two types of systems with respect to substrates and products, and intermediates involved, will be complementary to developing a deeper understanding of these oxygenative processes. The proposed studies will employ rapid kinetics spectroscopy, chemical quenching, and other enzymological methods. X-ray crystallography and genetic techniques, including cloning, expression, and mutagenesis, will also be used to develop a better understanding of how the proteins catalyze these interesting reactions. Our approach will be to modify active site residues, and then to study by a variety of physical techniques how various steps in catalysis are affected.We believe that results from these studies will lead to a better understanding of how molecular oxygen is activated for controlled metabolic processes. This may, in turn, lead to an improved ability to predict how various compounds will be metabolized in the environment.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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Preusch, Peter C
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Spolitak, Tatyana; Ballou, David P (2015) Evidence for catalytic intermediates involved in generating the chromopyrrolic acid scaffold of rebeccamycin by RebO and RebD. Arch Biochem Biophys 573:111-9
Singh, Sangita; Ballou, David P; Banerjee, Ruma (2011) Pre-steady-state kinetic analysis of enzyme-monitored turnover during cystathionine ?-synthase-catalyzed H(2)S generation. Biochemistry 50:419-25
Galinato, Mary Grace I; Spolitak, Tatyana; Ballou, David P et al. (2011) Elucidating the role of the proximal cysteine hydrogen-bonding network in ferric cytochrome P450cam and corresponding mutants using magnetic circular dichroism spectroscopy. Biochemistry 50:1053-69
Spolitak, Tatyana; Funhoff, Enrico G; Ballou, David P (2010) Spectroscopic studies of the oxidation of ferric CYP153A6 by peracids: Insights into P450 higher oxidation states. Arch Biochem Biophys 493:184-91
Mayfield, Jeffery A; Frederick, Rosanne E; Streit, Bennett R et al. (2010) Comprehensive spectroscopic, steady state, and transient kinetic studies of a representative siderophore-associated flavin monooxygenase. J Biol Chem 285:30375-88
Tarasev, Michael; Pullela, Sailaja; Ballou, David P (2009) Distal end of 105-125 loop--a putative reductase binding domain of phthalate dioxygenase. Arch Biochem Biophys 487:10-8
Lee, Moon N; Takawira, Desire; Nikolova, Andriana P et al. (2009) Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli. Biochemistry 48:7673-85
Shebley, Mohamad; Kent, Ute M; Ballou, David P et al. (2009) Mechanistic analysis of the inactivation of cytochrome P450 2B6 by phencyclidine: effects on substrate binding, electron transfer, and uncoupling. Drug Metab Dispos 37:745-52
Jaganaman, Sunil; Pinto, Alex; Tarasev, Michael et al. (2007) High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli. Protein Expr Purif 52:273-9
Tarasev, Michael; Pinto, Alex; Kim, Duke et al. (2006) The ""bridging"" aspartate 178 in phthalate dioxygenase facilitates interactions between the Rieske center and the iron(II)--mononuclear center. Biochemistry 45:10208-16