STEROIDOGENIC P450 ENZYMES--STRUCTURAL MUTAGENESIS

类固醇激素的生物合成涉及细胞色素的几个成员 P450超家族，线粒体型和微粒体型。从长远来看 GM37942的目标仍然是阐明结构/功能 这些关键酶与重要生物的关系 流程。这次竞争更新主要集中在两种类固醇上 羟基酶，线粒体胆固醇侧链的裂解 细胞色素P450(P450scc)与微粒体17α-羟基酶 细胞色素P450(P45Oc17)。 Coli.具体目标是基于已公布和未公布的结果 在本授权期内取得。1)利用大肠杆菌 黄曲霉毒素作为含FMN的P450相互作用结构域的模型 P450还原酶，牛P450c17上的氨基酸残基 还原酶的相互作用将通过化学修饰和 定点突变。2)随机嵌合体发生将在E. Coli在共享共同的不同P450之间产生嵌合体 底物，但在不同的位置催化反应：牛 P450scc/人P450c27(胆固醇)；牛P450c17/大鼠P450c17 黄体酮；牛P450c17/牛P450c21(黄体酮) P450scc/大鼠P450c7(胆固醇)。从这些嵌合体区域需要 对于酶的特异性将被鉴定，这可以进行更多的分析 谨慎地进行定点突变。3)三级结构 牛肾上腺素将从现有的晶体中确定，而 P450scc和肾上腺素-P450scc复合体将一次测定 得到了晶体。对P450scc的初步研究导致了 结晶的起点很有希望。类似的战略将是 适用于P450c17和P450c17-黄曲霉毒素的结晶 很复杂。4)P450c17与辅助蛋白细胞色素的相互作用 B5和3-羟基类固醇脱氢酶，将被研究。网络电话公司。B5 改变人和牛的P450c17活性，3beta-HSD在 与P450c17和P450c21的联系提示存在复合体 在内质网中，这与生理相关。此外 为了提供有关P450scc和P450c17的详细信息，这些研究 将提供对结构/功能关系的总体洞察 线粒体和微粒体P450s。因为P450scc和P450c17玩 在调节血压、生殖、生长和 压力；这些研究将提供关于这些方面的新的生化见解 基本的生物过程。

Steroid hormone biosynthesis involves several members of the cytochrome P450 superfamily, both mitochondrial and microsomal forms. The long term goal of GM37942 continues to be elucidation of the structure/function relationships of these key enzymes involved in important biological processes. This competitive renewal focuses primarily on two steroid hydroxylases, the mitochondrial cholesterol side chain cleavage cytochrome P450 (P450scc) and the microsomal 17alpha-hydroxylase cytochrome P450 (P45Oc17), utilizing recombinant enzymes produced in E. coli. The Specific Aims are based on published and unpublished results obtained during the present granting period. 1) Utilizing E. coli flavodoxin as a model for the FMN-containing, P450-interacting domain of P450 reductase, amino acid residues on bovine P450c17 required for P450 reductase interaction will be identified by chemical modification and site directed mutagenesis. 2) Random chimeragenesis will be used in E. coli to generate chimeras between different P450s which share common substrates but catalyze reactions at different sites: bovine P450scc/human P450c27 (cholesterol); bovine P450c17/rat P450c17 (progesterone); bovine P450c17/bovine P450c21 (progesterone); bovine P450scc/rat P450c7 (cholesterol). From these chimeras regions required for enzymatic specificity will be identified, which can be analyzed more carefully by site directed mutagenesis. 3) The tertiary structure of bovine adrenodoxin will be determined from existing crystals, while that of P450scc and the adrenodoxin-P450scc complex will be determined once crystals are obtained. Preliminary studies on P450scc have led to a promising starting point for crystallization. Similar strategies will be applied to crystallization of both P450c17 and the P450c17-flavodoxin complex. 4) Interaction of P450c17 with accessory proteins, cytochrome b5 and 3-hydroxysteroid dehydrogenase, will be investigated. Cyt. b5 alters human and bovine P450c17 activities and 3Beta-HSD was purified in association with P450c17 and P450c21 suggesting existence of a complex in endoplasmic reticulum which has physiological relevance. In addition to providing detailed information on P450scc and P450c17, these studies will provide general insight into structure/function relationships of mitochondrial and microsomal P450s. Because P450scc and P450c17 play important roles in regulation of blood pressure, reproduction, growth and stress; these studies will provide new biochemical insights on these essential biological processes.