P450s are a large superfamily of proteins that are present in most every organism from bacteria to mammals, and from yeast to plants. In mammals, P450s function to metabolize xenobiotics, synthesize steroid hormones and prostaglandins, and more recently found to affect/modulate ion transport in kidney and heart with P450 arachidonic acid metabolites. There have been several prokaryotic P450s and one eukaryotic P450 which have been crystallized and their structures determined. Of these, P450-BMP, the P450 domain of a naturally occurring fusion protein between a P450 and its cognate NADPH reductase, is one of the best models for studying P450s. BMP is easily purified in large quantities and it falls into the same P450 structural subclass as the eukaryotic P450. In the past, we have determined the structure of the co-crystallization between the P450 domain with the FMN domain of the reductase, and recently we have crystallized BMP with novel substrates and collected X-ray data on two of these at a 1.75 A resolution. We have also constructed mutants with altered substrate binding and specificity, and studied electron transfer between the reductase and P450. Now we want to probe even deeper in our understanding of the structure and function of P450BM-3 and the superfamily of P450s. We want to define the structural determinants that control substrate recognition and binding, as well as the movement of the substrate on reduction of the heme iron. We will continue our mutagenesis studies of BMP in order to regio- and stereoselectively monooxygenate long chain fatty acids to produce compounds that have important biological activities. We will further construct P450 proteins with modified hemes for use in mechanistic studies of electron transfer, protein/protein interactions, and oxygen activation. Finally, we will continue to refine our tools for structural analysis, prediction, and molecular modeling of the P450 family.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Okita, Richard T
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University of Texas Sw Medical Center Dallas
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Davydov, Dmitri R; Ponomarev, Gelii V; Bobrovnikova-Marjon, Ekaterina et al. (2013) Aluminum-substituted heme domain of P450BM-3 (BMP): introducing a heme-derived fluorescent probe for studies of substrate binding and protein-protein interactions in cytochromes P450. Biotechnol Appl Biochem 60:41-51
Haines, Donovan C; Hegde, Amita; Chen, Baozhi et al. (2011) A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation. Biochemistry 50:8333-41
Haines, Donovan C; Chen, Baozhi; Tomchick, Diana R et al. (2008) Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel. Biochemistry 47:3662-70
Kitazume, Tatsuya; Haines, Donovan C; Estabrook, Ronald W et al. (2007) Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3. Biochemistry 46:11892-901
Hegde, Amita; Haines, Donovan C; Bondlela, Muralidhar et al. (2007) Interactions of substrates at the surface of P450s can greatly enhance substrate potency. Biochemistry 46:14010-7
Chowdhary, Puneet K; Keshavan, Neela; Nguyen, Hien Q et al. (2007) Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines. Biochemistry 46:14429-37
Mast, Natalia; Murtazina, Dilyara; Liu, Hong et al. (2006) Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies. Biochemistry 45:4396-404
Mast, Natalia; Graham, Sandra E; Andersson, Ulla et al. (2005) Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis. Biochemistry 44:3259-71
Conley, Alan; Mapes, Samantha; Corbin, C Jo et al. (2002) Structural determinants of aromatase cytochrome p450 inhibition in substrate recognition site-1. Mol Endocrinol 16:1456-68
Graham, Sandra E; Peterson, Julian A (2002) Sequence alignments, variabilities, and vagaries. Methods Enzymol 357:15-28

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