P450BM 3--A MODEL FOR EUKARYOTIC P450S

P450BM 3--真核P450S模型

• 批准号: 2188993
• 负责人: JULIAN A PETERSON
• 金额: $ 14.46万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188993
• 关键词: active sites; bacterial proteins; chemical binding; computer system hardware; cytochrome P450; enzyme mechanism; enzyme structure; enzyme substrate; eukaryote; gas chromatography; membrane proteins; microorganism culture; nucleic acid sequence; oxidation; potentiometry; site directed mutagenesis; spectrometry; stereochemistry; stop flow technique; thermodynamics

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?

超家族中有超过两百个已测序的成员 蛋白质P450 在细菌，真菌， 植物和动物 在哺乳动物中，这些酶是极其重要的 在药物解毒、类固醇生成和致癌作用中， 有些细菌参与有机化合物的同化作用。 P450单加氧酶可以根据其组成分为两类。 所需的氧化还原伴侣：I类P450，需要铁硫蛋白 和还原酶存在于细菌和 真核生物和II类P450，仅需要含有FAD/FMN的 还原酶主要以膜结合的微粒体蛋白形式存在 在真核生物中。 直到最近，唯一已知的X射线晶体结构 a P450是P450 cam的P450，P450 cam是一种可溶性I类P450， 樟脑 现在，这个研究小组已经确定了两个 更多的P450：另一种可溶性细菌I类P450，P450 terp， 重要是，可溶性细菌II类P450，P450 BM-P. P450 BM-P， 天然存在的融合蛋白P450 BM-3的P450结构域，其 含有P450结构域和还原酶结构域。 P450cam，P450terp， 和P450 BM-3是不同基因家族的成员， 不同的生理底物。 的三维结构 这些蛋白质虽然大体相似，但不同，特别是在 参与底物和氧化还原伴侣结合的区域。 因此，虽然 P450 cam和P450 terp是I级P450的良好型号，P450 BM-3是 唯一已知的II级P450型号，因此， 了解的机制和蛋白质结构所需的 真核微粒体P450中的电子传递。 此外，本发明还 由于所有三种的基板是不同的，P450 cam和 P450 terp是非常特异的，P450 BM-P是脂肪酸 单加氧酶将氧化各种其他化合物， 控制氧化特异性的结构特征可以 研究了 最后，底物进入P450 cam中的活性位点 从它的三维结构来看并不明显;然而， P450 BM-P的结构中存在一个开放的通道，适合研究 底物识别和进入活性位点的能力。 为此， 本研究计划将集中于以下具体目标：A. 催化氧化反应的立体和区域特异性 P450 BM-3通过合理重新设计活性位点而改变？ B。什么 疏水底物识别区的作用，在口 基板存取通道，用以控制基板 P450 BM-P的特异性？ C.这种运动和相互作用 在P450 BM的富含β-折叠和富含α-螺旋的结构域之间， P控制衬底进入衬底进入通道，以及 因此，到活性位点？ D.是P450与其 由“真核生物”控制的生理相关氧化还原配偶体 插入序列，其包括J'螺旋和两个3/10螺旋 在K'螺旋之后;并且是假定的氧化还原伴侣的变化 对接区域能够改变氧化还原伙伴的特异性？ E. P450 BM-P能否从可溶性蛋白质转变为膜结合蛋白质 以及这将如何影响其底物结合及其与 氧化还原伙伴？