Factors that control the process of molecular recognition: protein folding and cofactor binding; and, substrate are the focus of our research on the P450 enzyme system. P450s are ubiquitous in the aerobic biosphere where they perform functions that are vital to life. There have been estimates that there are over 200 P450s in the human genome. The reactions catalyzed by P450s usually require an external source of reducing equivalents, NAD(P)H, and auxiliary proteins to transfer the electrons to the P450. The P450 gene superfamily can be divided into classes based on the mode of delivery of electrons from NAD(P)H to the P450. We are currently studying two structurally defined P450s. We isolated, purified, cloned, and sequenced the operon for a P450 from a Pseudomonad sp. which was obtained by culture enrichment techniques for its ability to grow on a -terpineol, a bacteriostatic, natural product. We have over expressed this protein in E. coli. thus providing us with the quantity of protein necessary for biophysical characterization. The other P450 which we are currently studying is P450BM-3, a bacterial homolog of eukaryotic microsomal P450s, which is a catalytically self-sufficient fatty acid w-hydroxylase/epoxidase. P450BM-3 has a molecular mass of 120,000 with both a flavoprotein reductase and a hemoprotein P450 domain on the same polypeptide chain. We have succeeded in subcloning, overexpressing in E. coli and purifying to homogeneity the P450 and reductase domains as well as the FAD- and FMN-containing sub-domains of the reductase domain. In collaboration with Dr. Hans Deisenhofer’s research group, we have determined the atomic structure of both P450terp and the hemoprotein domain of P450BM-3. Thus, we are in an excellent position to utilize these proteins to explore substrate recognition and binding, protein-protein interaction and electron transfer, protein folding, and the structural determinants which make a protein a member of the P450 gene superfamily. In pursuit of these aims, we employ a variety of biochemical and biophysical tools appropriate to the task rather than focusing on a single methodology. Recently we have employed rapid reaction techniques to study inter- and intra-domain electron transfer, site directed mutagenesis to study substrate binding and metabolism by the P450 domain, and, homology modeling to construct models of eukaryotic P450s based on the structure of P450BM-3.
RESEARCH INTERESTS
Mechanism of enzymatic oxygen activation
Electron Transport
Protein-protein interaction
Enzyme kinetics
Site-directed mutagenesis and protein engineering
RECENT PUBLICATIONS
Haines, D. C., Tomchick, D. R., Machius, M., and Peterson, J. A., ""The pivotal role of water in the mechanism of P450BM-3"" Biochemistry, 40:13456-13465, 2001
Falck, J. R., Reddy, Y. K., Haines, D. C., Reddy, K. M., Krishna, U. M., Graham, S. E., Murry, B., and Peterson, J. A., ""Practical, enantiospecific synthesis of 14,15-EET and leukotoxin B (vernolic acid)"" Tetrahedron Lett., 42:4131-4133, 2001
Daiber, A., Herold, S., Schoneich, C., Namgaladze, D., Peterson, J. A., and Ullrich, V., ""Nitration and inactivation of cytochrome P450(BM-3) by peroxynitrite-Stopped-flow measurements prove ferryl intermediates"" Eur. J. Biochem., 267:6729-6739, 2000
Kariakin, A., Davydov, D. R., Peterson, J. A., and Jung, C., ""A new approach to the study of protein-protein interaction by FTIR: Complex formation between cytochrome P450BM-3 heme domain and FMN reductase domain"" Biochemistry, 41:13514-13525, 2002
Usanov, S. A., Graham, S. E., Lepesheva, G. I., Azeva, T. N., Strushkevich, N. V., Gilep, A. A., Estabrook, R. W., and Peterson, J. A., ""Probing the interaction of bovine Cytochrome P450scc (CYP11A1) with adrenodoxin: Evaluating site-directed mutations by molecular modeling"" Biochemistry, 41:8310-8320, 2002
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