This project is a multi-disciplinary study of the structure and function of oxygenases and other redox enzymes. Our goal is to understand the chemical mechanism whereby oxygen is activated by oxygenases. 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. These enzymes are crucial to a majority of life forms.
The aims of this proposal are to investigate four different types of oxygenases which we have isolated in homogeneous form. We will elucidate intermediates in the reactions and define how amino acid residues affect functions of the following proteins: 1) Flavoprotein hydroxylases such as para-hydroxybenzoate hydroxylase. 2) Phthalate dioxygenase, a multicomponent dioxygenase system which converts an unactivated aromatic compound to a dihydrodiol. This type of oxygenase is very important in environmental degradation of aromatic compounds, and is poorly understood at present. 3) A collaborative study with S. Ragsdale on acetyl-CoA biosynthesis in anaerobic bacteria. This system consists of a series of enzymes with cobalamin, nickel, and iron-sulfur centers. The reactions involve methyl transfers to and from cobalamin, very similar to those we have measured with methionine synthase. 4) Galactose oxidase, a copper containing enzyme with an unusual tyrosine radical. We will investigate the participation of this radical in the oxidation of numerous glycolic substrates. The proposed study will employ rapid kinetics spectrophotometry, chemical quenching, and other enzymological methods. X-ray crystallography and genetic techniques, including cloning, gene sequencing and mutagenesis, will be used extensively to develop a better understanding of these interesting enzymes. Our approach will be to modify active site residues that activate the substrate or cofactors and then to study by the above physical techniques how various steps in catalysis, including formation and reactivity of intermediates, are affected. We hope that results from these studies will lead to a better understanding of how molecular oxygen is activated for controlled metabolic processes. This in turn may lead to the ability to predict how various compounds will be metabolized in the environment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM020877-24
Application #
2838439
Study Section
Special Emphasis Panel (NSS)
Project Start
1978-09-01
Project End
2001-11-30
Budget Start
1998-12-01
Budget End
1999-11-30
Support Year
24
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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
Tarasev, Michael; Kaddis, Catherine S; Yin, Sheng et al. (2007) Similar enzymes, different structures: phthalate dioxygenase is an alpha3alpha3 stacked hexamer, not an alpha3beta3 trimer like ""normal"" Rieske oxygenases. Arch Biochem Biophys 466:31-9