Redox enzymes participate in all organisms and are crucial to normal metabolism of endogenous substrates, and for the biosynthesis and biodegradation of pharmacological and toxic substances. We are examining flavoprotein, hemoprotein, and nonheme iron oxygenases, as well as other redox proteins that can be isolated in homogeneous form with the goal of elucidating fundamental mechanisms of enzymological catalysis. In particular, we study how several of these enzymes activate oxygen and control reactions with organic compounds. Our investigations use a variety of approaches, including spectral and rapid reaction techniques for characterizing active sites and intermediates in the reactions, site-directed mutagenesis to specifically test roles of particular amino acid residues in each of the steps of the reaction, and X-ray crystallography, in collaboration with other laboratories, to define the relationship of structure and function in these enzymes and their mutants
I. Flavoprotein oxygenases carry out hydroxylations of many substances including aromatic compounds. Some of these enzymes use both a flavin reductase and an oxygenase, and others use a single protein to carry out these complex reactions. We are studying how the protein environments efficiently bring about catalysis of hydroxylations, halogenations, and even flavin cannibalism, reactions that are rarely accomplished by organic flavin models. We have active collaborations with Dr. Barrie Entsch in Australia, Dr. Pimchai Chaiyen in Thailand, Dr. Chris Walsh at Harvard, and Dr. Graham Walker at MIT.
II. Nonheme iron-containing dioxygenases and monooxygenases in soil bacteria are critical to the microbial degradation of persistent and toxic aromatic and aliphatic compounds in the environment. We are investigating electron transfer reactions between the FMN and [2Fe–2S] centers and the mechanism of the oxygenation reaction at the mononuclear iron center of the oxygenase of the 2-component phthalate dioxygenase system. This system is a prototype for Rieske nonheme iron oxygenases that catalyze oxygenations of poorly reactive aromatic compounds.
III. We are investigating higher oxidations states of cytochromes P450 to better determine their potential in oxygenating a large variety of compounds including endogenous and xenobiotic chemicals. P450s are especially important in drug metabolism. In addition, many cytochrome P450s are involved in the biosynthesis of pharmaceutically important compounds. We are using hydroperoxides, peracids, and other oxidants to generate higher oxidations states of P450, including Compound I (oxidation state of +5), which contains ferryl (Fe=O) and a porphyrin cationic radical, Compound II (oxidation state of +4), which contains ferryl, and Compound ES (oxidation state of +5), which contains ferryl and an amino acid radical. These studies are in collaboration with Dr. John Dawson at the University of South Carolina, and Dr. Brian Hoffman at Northwestern University.
Other Redox Enzymes
IV. We are studying thioredoxin reductase and related flavoprotein dithiol reductases in collaboration with laboratories in Germany, Scotland, and Rush Medical School. This project is co-directed by Dr. Charles Williams, Jr. These enzymes are crucial to many biological functions, especially those dealing with oxidative stress and protein folding. We are investigating enzymes from human, mosquito, and the malarial causative agent P. falciparum with the hope of developing inhibitory drugs for combating malaria, and from schistosomes with the aim of finding drugs to combat schistosomiasis.
V. We are also collaborating with several laboratories on studies of additional redox enzymes. Among these are: James Bardwell (U of Michigan) on proteins using disulfide-dithiol interchange mechanisms that aid in protein folding, Paul Hollenberg (U of Michigan) on P450 enzymes, and Neil Marsh (U of Michigan) on glutamate mutase (B12), and Elizabeth Trimmer (Grinnell College) on the flavoprotein, methylene tetrahydrofolate reductase.
When Dave is not doing biochemistry, he is never without things to do. He likes to mountain bike, ski (downhill and cross-country), sail, dance, listen to and play music, and play tennis and softball. He is married to Jean Ballou and they have four children, and seven grandchildren.
PubMed Search Term: ballou dp
Recent Representative Publications from the Ballou Laboratory
Flavin Dependent Hydroxylases
1. Entsch, B., Cole, L J., Ballou, D. P., Protein dynamics in the function of p-hydroxybenzoate hydroxylase. Arch. Biochem. Biophys., 2005, 433, 297-311.
2. Cole, L. J., Gatti, D. L., Entsch, B., Ballou, D. P., Removal of a Methyl Group Causes Global Changes in p-Hydroxybenzoate Hydroxylase. Biochemistry, 2005, 44, 8047-8058.
3. Sucharitakul, J., Chaiyen, P., Entsch, B., and Ballou, D. P., The reductase of p-hydroxy-phenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis. Biochemistry, 2005, 44, 10434-10432.
4. Cole, L. J, Entsch, B., Ortiz-Maldonado, M., and Ballou, D. P,. Properties of p-Hydroxybenzoate Hydroxylase When Stabilized in Its Open Conformation. Biochemistry, 2005, 44 14807-14817.
5. Ballou, D. P., Entsch, B., Cole, L. J., Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases. Biochem. Biophys. Res. Commun. 2005, 338, 590-598.
6. Brender, J. R., Dertouzos, J., Ballou, D. P., Massey, V., Palfey, B. A., Entsch, B., Steel, D. G., Gafni, A. Conformational Dynamics of the Isoalloxazine in Substrate-free p-Hydroxybenzoate Hydroxylase: Single-Molecule Studies. J. American Chemical Society, 2005, 127, 18171-18178.
7. Sucharitakul, J., Chaiyen, P., Entsch, B., and Ballou, D. P., Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii. J. Biol. Chem., 2006, 281, 17044-17053.
8. Yeh, E., Cole, L. J., Barr, E. W, Bollinger, J. M. Jr., Ballou, D. P., and Walsh, C. T., Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH. Biochemistry, 2006, 45, 7904-7912.
9. Sucharitakul J, Phongsak T, Entsch B, Svasti J, Chaiyen P, Ballou D, P., Kinetics of a Two-Component p-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the Oxygenase. Biochemistry 2007, 46, 8611-8623.
10. Ballou D. P., Crystallography gets the jump on the enzymologists. Proc Natl Acad Sci U S A. 2007, 104, 15587-8.
11. Suadee C, Nijvipakul S, Svasti J, Entsch B, Ballou D. P., Chaiyen P. Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues. J Biochem (Tokyo). 2007, 142, 539-552.
12. Nijvipakul, S., Wongratana, J., Suadee, C., Entsch, B., Ballou, D.P., Chaiyen, P., LuxG is a functioning flavin reductase for bacterial luminescence. J. Bacteriol. 2008, 190, 1531-1538.
13. Valton, J., Mathevon, C., Fontecave, M., Nivière, V., and Ballou, D. P., The Two-component FMN-Dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor: Mechanism and Regulation J. Biol. Chem. 2008, 283, 194-196.
14. Entsch, B. and Ballou, D. P., Flavin-Mediated Hydroxylation Reactions, in Wiley Encyclopedia of Chemical Biology (in press).
1. Spolitak, T., Dawson, J. H., Ballou, D. P., Reaction of ferric cytochrome P450CAM with peracids: Kinetic characterization of intermediates on the reaction pathway. J Biol Chem. 2005, 280, 20300-20309.
2. Glascock, M.C., Ballou, D. P., and Dawson, J. H.. Effector Role of Reduced Putidaredoxin on the Oxygenation Reaction of Cytochrome P450-CAM; Perturbed Oxyferrous Form, a New Intermediate in the Reaction. J. Biological Chemistry, 2005, 280, 42134-42141.
3. Raner, G.M., Thompson, J. I., Haddy, A.,Tangham, V., Bynum, N., Reddy, R., Ballou, D. P., Dawson, J. H.,, Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors.. J. Inorg. Biochem. 2006, 100, 2045-53.
4. Spolitak, T., Dawson, J. H., and Ballou, D. P., Rapid Kinetics Studies of Peracid Oxidation of Ferric P450cam: Nature and Possible Function of Compound ES J. Inorg. Biochem. 2006, 100, 2034-44.
5. Spolitak, T., Dawson, J.H., Ballou, D. P., Replacement of Tyrosine Residues by Phenylalanine in Cytochrome P450cam Alters Formation of Cpd II-like Species in Reactions with Artificial Oxidants J. Biol Inorg. Chem, 2008, 13, 599-611.
Nonheme Iron Oxygeneses
1. Tarasev, M., Rhames, F., Ballou, D. P., Rates of Phthalate Dioxygenase Reaction with Oxygen are Dramatically Increased by Interactions with Phthalate and Phthalate Dioxygenase Reductase. Biochemistry 2004, 43, 12799-12808.
2. Grzyska, P. K., Ryle, M.J., Monterosso, G. R., Liu, J., Ballou, D. P., Hausinger, R. P., Steady-State and Transient Kinetic Analyses of Taurine/alpha-Ketoglutarate Dioxygenase: Effects of Oxygen Concentration, Alternative Sulfonates, and Active-Site Variants on the Fe(IV)-oxo Intermediate. Biochemistry 2005, 44, 3845-3855.
3. Tarasev, M., Ballou, D. P., Chemistry of the Catalytic Conversion of Phthalate into Its cis-Dihydrodiol during the Reaction of Oxygen with the Reduced Form of Phthalate Dioxygenase. Biochemistry, 2005, 44, 6197-61207.
4. Pinto, A, Tarasev, M., and Ballou, D. P., Substitutions of the Bridging Aspartate 178 Result in Profound Changes in the Reactivity of the Rieske Center of Phthalate Dioxygenase. Biochemistry, 2006, 45, 9032-9041.
5. Tarasev, M., Pinto, A, Duke Kim, Sean J. Elliott, and Ballou, D. P., The Bridging Aspartate 178 in Phthalate Dioxygenase Facilitates Interactions between the Rieske Center and the Fe(II)-Mononuclear center. Biochemistry, 2006, 45, 10208-10216.
6. Jaganaman S., Pinto A., Tarasev M., Ballou D. P., High levels of expression of the iron-sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli. Protein Expr Purif. 2006, 52, 273-279.
7. Tarasev, M., Kaddis, C.S., Yin, S., Loo, J.A., Burgner, J., Ballou, D.P., Similar enzymes, different structures: phthalate dioxygenase is an alpha-3alpha3 stacked hexamer, not an alpha3-beta3 trimer like "normal" Rieske oxygenases. Arch Biochem Biophys. 2007, 466, 31-9.
1. Deponte, M., Urig, S., Arscott, L. D., Fritz-Wolf, K., Reau, R., Herold-Mende, C., Koncarevic, S., Meyer, M., Davioud-Charvet, E., Ballou, D. P., Williams, C. H. Jr, Becker, K., Mechanistic studies of a novel, highly potent gold-phosphole inhibitor of human glutathione reductase, J Biol Chem. 2005, 280, 20628-20637.
2. Johansson, L., Arscott, D., Ballou, D. P., Williams, C. H. Jr., Arner, E. S. .J., Studies of an active site mutant of the selenoprotein thioredoxin reductase: The Ser-Cys-Cys-ser Motif of the Insect Orthologue is Not Sufficient to Replace the Cys-Sec Diad in the Mammalian Enzyme. Free Radicals in Biology and Medicine 41, 649-656.
3. McMillan, P. J., Arscott, L. D., Ballou, D. P., Becker, K., Williams, C. H. Jr., Muller, S. Identification of acid-base catalytic residues of high-MR thioredoxin reductase from plasmodium falciparum. J. Biol Chem. 2006, 281, 32967-77.
4. Tapley, T. L., Eichner, T., Gleiter, S., Ballou, D. P., Bardwell, J. C., Kinetic Characterization of the Disulfide Bond Forming Enzyme DsbB. J. Biol. Chem. 2007, 282, 10263-10271.
5. Cheng, Z., Arscott, L. D., Ballou, D. P., Williams, C. H. Jr., The relationship of the redox potentials of thioredoxin and thioredoxin reductase from Drosophila melanogaster to the enzymatic mechanism: reduced thioredoxin is the reductant of glutathione in Drosophila. Biochemistry 2007, 46, 7875-85.
6. Huang, H-H., Arscott, L. D., Ballou, D.P., and Williams, C.H. Jr., Acid-base catalysis in the mechanism of thioredoxin reductase from Drosophila melanogaster. Biochemistry 2008, 47, 1721-1731.
1. Trimmer, E. E., Ballou, D. P., Galloway, L, J., Scannell, S. A., Brinker, D. R., Casas, K. R., Aspartate 120 of Escherichia coli methylenetetrahydrofolate reductase: evidence for major roles in folate binding and catalysis and a minor role in flavin reactivity. Biochemistry, 2005, 44, 6809-6822.
2. Alexander, J. P., Ryan, T. J., Ballou, D. P., Coward, J. K., Gamma Glutamyl Hydrolase: Kinetic Characterization of Isopeptide Hydrolysis Using Fluorogenic Substrates. Biochemistry, 2008, 47, 1228-1239.
3. Padovani, D., Labunska, T., Palfey, B. A., Ballou, D. P., Banerjee, R., Adenosyltransferase tailors and delivers coenzyme B12. Nature: Chem. Biol. 2008, 4, 194-196.