P450BM-3--STRUCTURE AND BIOENGINEERING

P450是一个很大的蛋白质超家族，存在于从细菌到哺乳动物，从酵母到植物的几乎所有有机体中。在哺乳动物中，P450的功能是代谢外源物质，合成类固醇激素和前列腺素，最近发现通过P450花生四烯酸代谢产物影响/调节肾脏和心脏的离子转运。目前已有多个原核生物P450和一个真核生物P450已经结晶并确定了它们的结构。其中，P450-BMP是P450与其同源NADPH还原酶之间自然产生的融合蛋白的P450结构域，是研究P450的最佳模型之一。BMP易于大量纯化，属于与真核细胞P450相同的结构亚类。在过去，我们已经确定了P450结构域和还原酶的FMN结构域之间的共结晶结构，最近我们用新的底物结晶了BMP，并收集了其中两种底物的1.75A分辨率的X射线数据。我们还构建了底物结合和特异性改变的突变体，并研究了还原酶和P450之间的电子转移。现在，我们想要更深入地了解P450BM-3和P450超家族的结构和功能。我们想定义控制底物识别和结合的结构决定因素，以及底物在还原血红素铁时的运动。我们将继续BMP的诱变研究，以区域和立体选择性地单氧化长链脂肪酸，生产具有重要生物活性的化合物。我们将进一步用修饰的氯化血红素构建P450蛋白，用于电子转移、蛋白质/蛋白质相互作用和氧激活的机制研究。最后，我们将继续改进我们的工具，用于P450家族的结构分析、预测和分子建模。

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.