P450BM-3--STRUCTURE AND BIOENGINEERING

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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.