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Gregory M. Raner

Greg Raner
Title Associate Professor
Expertise Biochemistry, Bioinrganic
Education B.S., LeMoyne College , Syracuse NY , 1986
M.S., Syracuse University Syracuse , NY , 1989
PhD., University of Utah , Salt Lake City , UT , 1993
Postdoctoral Fellow, University of Michigan, 1993-1997
Office Sullivan Science Building , Rm 416
Phone 336.334.4519
E-Mail Email Dr. Raner

RESEARCH INTERESTS

My research is mostly centered around a single class of enzymes, the cytochrome P450s.  These enzymes are present in nearly all life forms ranging from simple one-celled organisms to humans, and catalyze oxidative or reductive reactions in support of a variety of cellular functions.  In humans, one of the primary functions is the removal of foreign chemicals (including many pharmaceutical agents) from the body.  The enzymes use molecular oxygen and the cellular reducing agent NADPH to oxidize the foreign compounds which usually facilitates their elimination by a supporting class of enzymes called Phase II enzymes.

Project I:   Stopped-Flow Spectrophotometric Analysis of Reactive Cytochrome P450 Species. 
Stopped-Flow is a technique that allows for rapid mixing of two reacting species and monitoring of the progress of the reaction using electronic absorption within a few milliseconds of mixing.It is a very useful method for observing transient intermediates that may occur in the course of an enzyme catalyzed reaction.Consequently, much can be learned about the chemical mechanism of an enzyme by directly observing intermediate species formed during catalysis.Several projects in my lab are designed to use the stopped-flow technique to monitor the reaction of cytochrome P450BM3 with reagents that can transfer an oxygen atom to the metal center of the enzyme, thereby generating an enzyme intermediate in a highly oxidized state.This highly oxidized state is considered to be important in the overall mechanism of cytochrome P450-catalyzed reactions and characterization of observed spectral intermediates would provide insight into the chemical mechanism utilized by this class of enzyme.We are using site-directed mutagenesis to prepare mutants of P450BM3 in which active site residues are specifically modified in a further attempt to characterize and identify certain intermediates that have been observed.

Project II:Carbon-13 labeling of the heme cofactor of cytochrome P450 and peroxidase enzymes.  The selective incorporation of carbon-13 into the heme group of several different heme containing enzymes has been accomplished using a strain of E.coli that has been engineered to block its ability to produce aminolevulenic acid, a biosynthetic heme precursor.  By synthesizing carbon-13 labeled aminolevulenic acid from glycine, it is possible to incorporate 13-C into the heme cofactor of any heme-containing enzyme that can be produced in E.coli. Studies involving paramagnetic NMR have been carried out with several heme-containing enzymes and information regarding electronic distributions and spin-densities within the heme macrocyle has been obtained on these systems. The methods used to prepare 13C-labeled heme in these studies involved heme removal and reconstitution of the protein being analyzed. Cytochrome P450 enzymes, by vitue of their heme-thiolate ligation, are very difficult to reconstitute, particularly at the concentrations required for NMR. The methods we have developed should open up a new technology for the study of cytochrome P450 enzymes.

Project III:   Interaction of Mammalian Cytochrome P450 Enzymes by Herbal Extracts, Essential oils, and their Constituents. In collaboration with the Cech Lab we have initiated a research project that is intended to elucidate potential herb drug or herb-xenobiotic interactions that may have significant health implications. Using human liver microsomes and various expressed human cytochrome P450 enzymes we have identified interactions between the human P450s and constituents in Goldenseal extract, Echinacea purpurea extract and Spilanthes acmella extracts. Attempts to isolate and characterize these constituents are underway and involve a variety of separations techniques and assay procedures for monitoring various P450 activities. We have also begun to look at essential oils and their ability to block the action of several human cytochrome P450 enzymes as a possible source of anti-oxidant properties of the oils. Along these lines, we have started to explore other potential anti-oxidant mechanisms associated with the use of various essential oils, and certain key constitutents

REPRESENTATIVE PUBLICATIONS

  1. Raner, G.M., A.D.N. Vaz, and M.J. Coon.Metabolism of All-trans, 9-cis, and 13-cis Isomers of Retinal by Purified Isozymes of Microsomal Cytochrome P450 and Mechanism-based Inhibition of Retinoid Oxidation by Citral.Mol. Pharmacol49:515-522 (1996).
  2. Vaz, A.D.N., S.J. Pernecky, G.M. Raner, and M.J. Coon. Peroxo-iron and Oxenoid-iron Species as Alternative Oxygenating Agents in Cytochrome P450-catalyzed Reactions: Switching by Threonine-302 to Alanine Mutagenesis of Cytochrome P450 2B4. Proc. Natl. Acad. Sci. U.S.A.93:4644-4648 (1996).
  3. Guengerich, F.P., A.D.N. Vaz, G.M. Raner, S.J. Pernecky, and M.J. Coon.Evidence for a Role of a Perferryl-Oxygen Complex, FeO3+, in the N-Oxygenation of Amines by Cytochrome P450 Enzymes.Mol. Pharmacol51:147-151 (1997).
  4. Raner, G.M., E.W. Chiang, A.D.N. Vaz, and M.J. Coon.Mechanism-Based Inactivation of Cytochrome P450 2B4 by Aldehydes:Relationship to aldehyde Deformylation via a Peroxyhemiacetal Intermediate. Biochemistry 36:4895-4902 (1997).
  5. Williams, S.K.R., G.M. Raner, W.R. Ellis, Jr., and J.C. Giddings.Separation of Protein Inclusion Bodies from Escherichia coliLysates Using Sedimentation Field-Flow Fractionation.J. Micro. Sep9:233-239 (1997).
  6. Raner, G.M., L.J. Martins, and W.R. Ellis, Jr. Functional Role of Leucine-103 in Myohemerythrin. Biochemistry 36:7037-7043 (1997).
  7. Zhang, Q.-Y., Raner, G., Ding, X., Dunbar, D., Coon, M.J., and Kiminsky, L.S.Characterization of CYP2J4:Expression in Rat Small Intestine and Role in Retinoic Acid Biotransformation from Retinal.Arch. Biochem. Biophys353:257-264 (1998).
  8. Shank-Retzlaff, M.L., G.M. Raner, M.J. Coon, and S.G. Sligar.The Membrane Topology of Cytochrome P450 2B4 in Langmuir-Blodgett Monolayers. Arch Biochem. Biophys359: 82-88 (1998).
  9. Kuo, C.-L., G.M. Raner, A.D.N. Vaz, and M.J. Coon.Discrete Species of Activated Oxygen Yield Different Cytochrome P450 Heme Adducts from Aldehydes.Biochemistry 38:10511-10518 (1999).
  10. Lloyd, C.R., G.M. Raner, A. Moser, E.M. Eyring, and W.R. Ellis, Jr.Oxymyohemerythrin:Discriminating Between O2 Release and Autoxidation.J. Inorg. Biochem. 81:293-300 (2000).
  11. Raner, G.M. A.J. Hatchell, P.E. Morton, D.P. Ballou, and M.J. Coon. Stopped-Flow Spectrophotometric Analysis of Intermediates in the Peroxo-Dependent Inactivation of Cytochrome P450 by Aldehydes. J. Inorg. Biochem81: 153-160 (2000).
  12. Raner, G.M., A.Q. Muir, C. Lowry and B.A. Davis. Farnesol as an Inhibitor and a Substrate for Rabbit Liver Microsomal P450 Enzymes. Biochem. Biophys. Res. Commun. 293, 1-6 (2002).
  13. Raner, G.M., J.A. Hatchell, M.U. Dixon, T. Joy, A.E. Haddy, and E.R. Johnston. Regioselective Peroxo-Dependent Heme Alkylation in P450BM3-F87G by Aromatic Aldehydes:  Effects of Alkylation on Catalysis.  Biochemistry 41, 9601-9610 (2002).
  14. Dixon, M.U., O. Omoseebi, A.A. Lawson, J. Bao, J.P. Handler, S.A. Brown, L. King, A. Mortenson, and G.M. Raner, Oxidation of 4-nitrophenol and 4-nitrocatechol by wild-type and mutant P450BM3 enzymes.  J. Undergrad. Chem. Res.  4, 149-155 (2002).
  15. Yang, S.-P., T. Medling, and G.M. Raner. Cytochrome P450 Expression and Activities in the Rat, Rabbit and Bovine Tongue. Comp. Biochem. Physiol. C. 136, 297-308 (2003).
  16. Yang, S.-P. and Raner, G.M.  Cytochrome P450 Expression, Induction and Activities in Human Tongue Cells and their Modulation by Green Tea Extract, Toxicol. Appl. Pharmacol. 202, 140-150 (2005).
  17. Perera, R., M. Sono, G.M. Raner, and J.H. Dawson. Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450BM3 (CYP102) oxygenase domain and holoenzyme. Biochem. Biophys. Res. Commun.  (2005).
  18. Cech, N. B., Tutor K. , Doty, B. , Spellman, K. , & Sasagawa, M. , Gregory M. Raner Cynthia A. Wenner.  Liver Enzyme-Mediated Oxidation of Echinacea purpurea Alkylamides: Production of Novel Metabolites and Changes in Immunomodulatory Activity.CHE Planta Medica, 72, 1372-1377 (2006).
  19. Raner, G. M., Thompson J. I., Haddy, A. , Tangham, V. , & Bynum, N. , Reddy Ramachandra David P. Ballou John H. Dawson,  Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors. J. Inorg. Biochem. 100 (12), 2045-2053 (2006).
  20. Osborne, R. L., Raner, G. M., Hager, L. P., & Dawson, J. H.  C. fumago chloroperoxidase is also a dehaloperoxidase: oxidative dehalogenation of halophenols. J. Amer. Chem. Soc., 128 (4), 1036-1037 (2006). 
  21. Yang, S., Wilson, K. , Kawa, A. , & Raner, G. M.  Effects of Green Tea Extracts on Gene Expression in HepG2 and Cal-27 Cells.CHE Journal of Food and Chemical Toxicology, 44, 1075-1081(2006).
  22. Reddick, J.J., Antolak, S.A., and Raner, G.M. PksS from Bacillus subtillus is a cytochrome P450 involved in bacillaene metabolism.  Biochem. Biophys. Res. Commun. 358:363-367 (2007).
  23. Raner, G.M., Cornelious, S., Moulick, K. Wang.Y., Mortenson, A. and Cech, N.B.  Effects of herbal products and their constituents on human cytochrome P4502E1 activity.  Food Chem. Toxicol. 45:2359-2365 (2007).
  24. Osborne, R.L., Coggins, M.K., Raner, G.M., Walla, M., Dawson, J.H. The mechanism of oxidative halophenol dehalogenation by Amphitrite ornata dehaloperoxidase is initiated by H2O2 binding and involves two consecutive one-electron steps: role of ferryl intermediates. Biochemistry48:4231-4238 (2009).
  25. Bryson, D, Lim, P.L., Lawson, A., Manjunath, S., and Raner, G.M.  Isotopic labeling of the heme cofactor in cytochrome P450 and other heme proteins.  Biotechnol. Lett. (In Press,  2011).