P450BM 3--A MODEL FOR EUKARYOTIC P450S

有超过200个已测序的超家族成员 一种叫做P450的蛋白质。它们已在细菌、真菌、 植物和动物。在哺乳动物中，这些酶极其重要 在药物解毒、类固醇合成和致癌方面，而在 一些细菌，它们参与有机化合物的同化。 P450单加氧酶可分为两类 必需的氧化还原伙伴：需要铁硫蛋白的I类P450 在细菌和沙门氏菌线粒体中发现了一种还原酶 真核生物，以及只需要含有FAD/FMN的II类P450 还原酶主要作为膜结合的微体蛋白被发现。 在真核生物中。直到最近，唯一已知的x射线晶体结构 P450是P450cam的P450，是一种可溶的I类P450，可羟化 樟脑。现在，这个课题组已经确定了两个 更多P450：另一个可溶性细菌I类P450、P450terp和大多数 重要的是，一种可溶性细菌II类P450，P450BM-P。P450BM-P为 天然存在的融合蛋白P450BM-3的P450结构域 含有P450结构域和还原酶结构域。P450摄像头，P450terp， 和P450BM-3是不同基因家族的成员，有三个 不同的生理底物。的三维结构 这些蛋白质虽然大体上相似，但却不同，特别是在 参与底物和氧化还原伙伴结合的区域。因此，虽然 P450cam和P450terp是I级P450的好机型，P450BM-3是 唯一已知的II类P450型号，因此，在 了解所需的机制和蛋白质结构 真核细胞微粒体P450的电子传递。另外， 由于这三种底物都不同，所以P450 cam和 P450terp是非常特异的，而P450BM-P是一种脂肪酸 会氧化多种其他化合物的单加氧酶， 控制氧化特异性的结构特征可以是 调查过了。最后，底物在P450摄像头中获得活性部位 从它的三维结构来看并不明显；然而，访问 P450BM-P结构中的通道是开放的，并服从于研究 底物识别和活性部位的访问。为了达到这些目的， 本研究项目将集中于以下具体目标：A. 氧化反应的立体专一性和区域专一性能否被催化 P450BM-3会被活性部位的合理重新设计所改变吗？B.什么？ 作用于疏水底物识别区域，位于口部 基板接入通道，起到控制基板的作用 P450BM-P？C.运动和相互作用 在P450BM的β-折叠富集域和α-螺旋富集域之间- P控制衬底进入衬底接入通道，以及 因此，到活动站点？D.是P450与其相互作用 “真核生物”控制的生理上相关的氧化还原伙伴 插入序列，包括J‘螺旋和两个3/10螺旋 跟随K‘螺旋；是假定的氧化还原伙伴的变化 对接区域能够改变氧化还原伙伴的专一性吗？E. P450BM-P能从可溶性蛋白质转变为膜结合形式吗 这将如何影响其底物结合和与其相互作用 氧化还原合伙人？

There are over two hundred sequenced members of the superfamily of proteins called P450. They have been identified in bacteria, fungi, plants, and animals. In mammals, these enzymes are extremely important in drug detoxification, steroidogenesis, and carcinogenesis, while in some bacteria they participate in the assimilation of organic compounds. P450 monooxygenases can be divided into two classes depending the required redox partner: class I P450s requiring an iron sulfur protein and a reductase are found in bacteria and in the mitochondria of eukaryotes, and class II P450s requiring only an FAD/FMN-containing reductase are found predominantly as membrane-bound, microsomal proteins in eukaryotes. Until recently the only known x-ray crystal structure for a P450 was that of P450cam, a soluble class I P450 which hydroxylates camphor. Now, this research group has determined the structure of two more P450s: another soluble bacterial class I P450, P450terp, and most importantly, a soluble bacterial class II P450, P450BM-P. P450BM-P is the P450 domain of the naturally occurring fusion protein P450BM-3 which contains both a P450 domain and a reductase domain. P450cam, P450terp, and P450BM-3 are members of different gene families and have three different physiological substrates. The three dimensional structures of these proteins, while generally similar, are different, especially in regions involved in substrate- and redox-partner binding. Thus, while P450cam and P450terp serve as good models for class I P450s, P450BM-3 is the only known model for a class II P450, and as such, is important in understanding the mechanism of and protein structure required for electron transfer in the eukaryotic microsomal P450s. Additionally, since the substrates of all three are different, with P450cam and P450terp being very specific, and P450BM-P being a fatty acid monooxygenase which will oxidize a variety of other compounds, the structural features which control the specificity of oxidation can be investigated. Finally, substrate access to the active site in P450cam is not apparent from its three-dimensional structure; however, the access channel is open in the structure of P450BM-P and amenable to the study of substrate recognition and access to the active site. To these ends, this research project will be focused on the following Specific Aims: A. Can the stereo- and regiospecificity of the oxidation reactions catalyzed by P450BM-3 be altered by rational redesign of the active site? B. What role does the hydrophobic substrate recognition region, at the mouth of the substrate access channel, play in controlling the substrate specificity of P450BM-P? C. Does the movement of and the interaction between the beta-sheet rich and the alpha-helical rich domains of P450BM- P control access of substrates into the substrate access channel, and hence, to the active site? D. Is the interaction of the P450 with its physiologically relevant redox partner controlled by the "eukaryotic" insertion sequences, which include the J' helix and the two 3/10 helices following the K' helix; and is a change of the putative redox-partner docking region able to alter the specificity for the redox partner? E. Can P450BM-P be converted from a soluble protein to a membrane-bound form and how will this effect its substrate binding and its interaction with redox partners?