P450BM3--STRUCTURE AND FUNCTION

p450 S被认为是严格的解毒/激素合成 在哺乳动物中的酶;然而，随着超过P450的鉴定， 哺乳动物，植物，真菌和细菌，已经意识到， P450的多样性比以前怀疑的要多得多。 与 P450 cam和我们的两个额外的P450的结构的阐明 实验室，P450 BM-P和P450 terp，我们已经能够确定 结构元件是高度保守的，哪些元件是较少的 保守的 这些P450在序列上具有极高的可变性 (less 10%以上）;然而，存在整体三维 结构，即类似于P450 cam，P450 terp， P450BM-P。 这个项目所围绕的假设是， P450结构折叠，因此，一个特定的折叠途径， 描述了这个蛋白质超家族。 理解本 结构折叠在修饰P450酶活性的努力中是至关重要的 用于生物修复和药物合成。 因此，我们的目标是 建立折叠、展开、血红素重组的条件， 以便能够改变底物特异性和氧化还原配偶体结合。 具体来说，为了检验我们的假设，我们提出了以下目标： A. 评价P450 BM-P中的折叠/解折叠途径 I. 变性条件的建立和随后的 复性 二. 用血红素和血红素衍生物制备P$BM-P以探测 底物/氧结合口袋和血红素环境。 B。 识别那些允许正确折叠的结构元件， 对于底物结合，以及对于氧化还原伴侣结合， I. 使用合成寡核苷酸合成P450 BM-P 二. 系统地消除结构元素，以构建一个“最小” P450 三. 用其他p450的结构元件替代构建 具有杂交活性的杂交蛋白 C. 确定那些p450形式的三维结构， 最感兴趣/最有用 I. 评价P450 BM-P与花生四烯酸底物结合的结构 或二十碳五烯酸 二. 结晶并确定那些杂交/突变P450的结构 这可能揭示了P450折叠途径的最小结构。 圆二色谱法将用于评估 在这些蛋白质的折叠和展开过程中， 磁性圆二色光谱将用于检查的变化， 血红素的配体状态

p450S have been thought of as strictly detoxification/hormone synthesizing enzymes in mammals; however, with the identification of more than P450s in mammals, plants, fungi, and bacteria, it has been realized that the role of P450s is much more diverse than had been previously suspected. With the elucidation of the structure of P450cam and of two additional P450s in our laboratory, P450BM-P and P450terp, we have been able to determine which structural elements are highly conserved and which elements are less conserved. These P450's have an extremely high variability in sequence (less than 10% identity); however, there is an overall three dimensional structure, i.e. a structural fold which is similar in P450cam, P450terp, and P450BM-P. The hypothesis around which this project has coalesced is that there is a P450 structural fold, and hence, a specific folding pathway which characterized this superfamily of proteins. The understanding of this structural fold is critical in the efforts to modify P450 enzyme activities to be used in bioremediation and drug synthesis. Therefore, our aims are to establish conditions for folding, unfolding, an heme reconstitution in order to be able to modify substrate specificity and redox partner binding. Specifically, to test our hypothesis we have set forth the following aims; A. Evaluating the folding/unfolding pathway in P450BM-P i. establishment of conditions for denaturation and subsequent renaturation ii. preparation of P$%)BM-P with heme and heme derivatives to probe the substrate/oxygen binding pocket and heme environment. B. Identifying those structural elements which allow for correct folding, for substrate binding, and for redox-partner binding i. synthesis of P450BM-P using synthetic oligonucleotides ii. systematic elimination of structural elements to construct a "minimal" P450 iii. substitution of structural elements from other p450 s to construct hybrid proteins with hybrid activities C. Determining the three dimensional structures of those p450 forms which are of greatest interest/utility i. evaluate the structure of P450BM-P bound to the substrates arachidonic or eicosapenteneoic acid ii. crystallize and determine the structure of those hybrid/mutant P450s which might shed light on the minimal structure of a P450 folding pathway. Circular dichroism spectroscopy will be used to assess the changes in tertiary structure during folding and unfolding of these proteins, while magnetic circular dichroism spectroscopy will be used to examine changes in the ligand state of the heme.