STEROIDOGENIC P450 ENZYMES--STRUCTURAL MUTAGENESIS

我们的长期目标是确定区域和特定的氨基酸 在类固醇生成P450中发挥关键作用， 底物特异性的羟基化反应。 在 大多数物种的肾上腺皮质有两个线粒体P450（P450 SCC和 P45011 β）和两种微粒体P450（P45017 α和P450 C21）。 参与糖皮质激素的胆固醇生物合成 （皮质醇）、盐皮质激素（醛固酮）和C19雄激素 性激素的前体 与大多数形式的P450一样， 内源性底物代谢，这些类固醇羟化酶证明 高度的底物特异性， 它们的结合和它们羟基化的立体特异性 反应. 我们将利用细菌中的诱变和表达， 检查这些酶的两种性质的要求。 我们有 开发了相对高水平表达功能性 P45017 α和P450 SCC在E.杆菌 因此，我们建议 研究这两种类固醇的特定诱变实验 在这种容易的表达系统中， 不仅评价酶的性质，而且评价光谱 突变体的特性（底物结合和血红素结合） 内切酶 能够很容易地检查这些光谱特性 细菌中的酶为我们提供了评估 突变的类固醇羟化酶的结构特性， 以及它们的酶性质。 我们还将开发E.杆菌 表达P45011 β和P450 C21的系统用于类似的 诱变实验 此外，我们将使用细菌 用于评估突变体的结构/功能关系的表达系统 来自具有17 α-羟化酶的个体的人P45017 α形式 缺陷 我们还将纯化和表征细胞溶质 支持P45017 α活性的细菌还原酶 细菌 最后，我们将开发纯化程序 来自细菌膜的P45017 α和P450 SCC用于详细的 生物物理研究和结晶。 这些研究将提供 关于结构/功能关系的详细信息 特别是肾上腺皮质类固醇羟化酶和一般的P450 肯定会拓宽我们对这个基因超家族的理解 产品.

Our long term goal is to identify regions and specific amino acids within the steroidogenic P450s which play key roles in the unique substrate specificities of their hydroxylation reactions. In the adrenal cortex of most species two mitochondrial P450s (P450scc and P45011Beta) and two microsomal P450s (P45017alpha and P450C21) are involved in the biosynthesis from cholesterol of glucocorticoids (cortisol), mineralocorticoids (aldosterone) and the C19 androgen precursors of the sex hormones. Like most forms of P450 involved in endogenous substrate metabolism, these steroid hydroxylases demonstrate a high degree of substrate specificity both with respect to the steroids which they bind and the stereospecificity of their hydroxylation reactions. We will utilize mutagenesis and expression in bacteria to examine the requirements for both properties of these enzymes. We have developed procedures for relatively high level expression of functional forms of P45017alpha and P450scc in E. coli. Accordingly we propose specific mutagenic experiments to investigate these two steroid hydroxylases in this facile expression system which readily permits not only evaluation of enzymatic properties but also spectral characteristics (substrate-binding and heme-binding) of the mutant enzymes. The ability to easily examine the spectral properties of these enzymes in bacteria provides us the opportunity to estimate the effect of mutagenesis on the structural properties of steroid hydroxylase, as well as on their enzymatic properties. We will also develop E. coli systems expressing P45011beta and P450C21 to be used in similar mutagenesis experiments. In addition we will use the bacterial expression system to evaluate structure/function relationships of mutant forms of human P45017alpha from individuals having 17alpha-hydroxylase deficiency. We will also purify and characterize the cytosolic bacterial reductase which supports P45017alpha activities in intact bacteria. Finally we will develop procedures for purification of P45017alpha and P450scc from bacterial membranes to be used for detailed biophysical studies and crystallization. These studies will provide detailed information on the structure/function relationships of the adrenocortical steroid hydroxylases in particular and P450s in general and surely will broaden our understanding of this superfamily of gene products.