PROSTAGLANDIN 19 AND 20 HYDROXYLATION BY CYTOCHROME P450

细胞色素 P450 使前列腺素 19 和 20 羟基化

• 批准号: 2406510
• 负责人: BETTIE SUE SILER MASTERS
• 金额: $ 21.3万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2406510
• 关键词: Escherichia coli; NADPH cytochrome c2 reductase; RNase protection assay; chimeric proteins; cytochrome P450; eicosanoid metabolism; electron spin resonance spectroscopy; enzyme activity; enzyme model; enzyme reconstitution; gene expression; hydroxylation; laboratory rabbit; liposomes; micelles; molecular chaperones; nitric oxide synthase; prostaglandins; site directed mutagenesis; transfection /expression vector; transmission electron microscopy

The focus of this research proposal is the determination of molecular and catalytic properties of four members of the CYP4A gene subfamily of cytochromes P450 which hydroxylate fatty acids and eicosanoids primarily or exclusively in the psi-position. The original member of this closely related (greater than 85% homologous at the amino acid level) subfamily was the P4504A4, simultaneously isolated in this laboratory from the lungs of pregnant rabbits and from Kusunose's laboratory from progesterone-treated male rabbits. Johnson, et al cloned and expressed three kidney cDNAs encoding lauric acid psi-hydroxylases now known as CYP 4A5, 4A6, and 4A7. A partial cDNA encoding CYP4A4 was reported by Kusunose's laboratory and the intact, full length cDNA was isolated jointly by Johnson's and masters' laboratories. Although the functions of these cytochromes P450 are unknown, a large literature is developing in which they are implicated in hemodynamic function by controlling vascular tone. Cerebral and renal microvessels are contracted by 20- hydroxyeicosatetraenoic acid (the psi-hydroxylated product of arachidonic acid) at concentrations of less than 10 -10M. Due to the high degree of sequence homology of these enzymes found in kidney and lung, their implication in hemodynamic control, and their degree of substrate specificity, the following Specific Aims are planned: 1) Expression of the CYP4A7 gene product will be pursued using other E. coli strains as well as other vectors, in the presence and absence of a plasmid encoding E. coli chaperonins (groEL and groES). 2) The reconstitution of these cytochromes P450 will be attempted using conventional sonicated lipid preparations and by incorporation into liposomes (lipid vesicles) prepared by various techniques, including the use of amphipathic detergents. 3) site-directed mutagenesis based upon identifying specificity determinants from chimeric constructs will be performed with each of the CYP4A enzymes (4A4-4A7) and mutants will be expressed in E. coli. 4) By homology-based modeling of the CYP4A enzymes using the Bacillus megaterium heme domain crystal structure, modules and regions of these enzymes likely to be involved in docking with NADPH-cytochrome P450 reductase, the structure of which has been obtained by the Kim and Masters laboratories, will be identified and subjected to module exchange or site-directed mutagenesis. 5) Fusion proteins containing one of the CYP4A subfamily members and NADPH- cytochrome P450 reductase and a module or modules from the constitutive nitric oxide synthases which mediate the Ca2+/calmodulin control of the latter enzymes will be constructed. Utilizing these approaches, the determinants of substrate specificities and catalytic efficiencies of the CYP4A enzymes will be determined.

本研究计划的重点是分子的测定 CYP4A基因亚家族的四个成员的和催化特性 细胞色素 P450 羟基化脂肪酸和类二十烷酸 主要或专门在 psi 位置。 这个原来的成员 密切相关（氨基酸水平同源性大于 85%） 亚科为P4504A4，本实验室同时分离 来自怀孕兔子的肺部和 Kusunose 的实验室 黄体酮治疗的雄性兔子。 Johnson等人克隆并表达 编码月桂酸 psi-羟化酶的三个肾脏 cDNA 现在称为 CYP 4A5、4A6 和 4A7。 报道了编码 CYP4A4 的部分 cDNA 由 Kusunose 实验室分离出完整的全长 cDNA 由约翰逊和大师实验室联合开发。 虽然功能 这些细胞色素 P450 的数量未知，大量文献正在开发中 其中它们通过控制参与血液动力学功能 血管张力。 脑和肾微血管收缩 20- 羟基二十碳四烯酸（psi-羟基化产物 花生四烯酸）浓度低于10 -10M。 由于 在肾脏中发现的这些酶具有高度的序列同源性 和肺，它们对血流动力学控制的影响以及它们的程度 底物特异性，计划实现以下具体目标：1) 将使用其他大肠杆菌来表达 CYP4A7 基因产物。 大肠杆菌菌株以及其他载体，在存在或不存在的情况下 编码大肠杆菌伴侣蛋白（groEL 和 groES）的质粒。 2) 的 将尝试使用以下方法重建这些细胞色素 P450 传统的超声脂质制剂并通过掺入 通过各种技术制备的脂质体（脂质囊泡），包括 使用两亲性洗涤剂。 3) 定点诱变 从嵌合构建体中鉴定特异性决定簇将是 用每种 CYP4A 酶 (4A4-4A7) 进行，突变体将 在大肠杆菌中表达。 4) 通过基于同源性的 CYP4A 建模 使用巨大芽孢杆菌血红素结构域晶体结构的酶， 这些酶的模块和区域可能参与对接 NADPH-细胞色素P450还原酶，其结构已被 由金和马斯特斯实验室获得，将被识别和 进行模块交换或定点诱变。 5）融合 含有 CYP4A 亚家族成员之一和 NADPH- 的蛋白质 细胞色素P450还原酶和来自于的一个或多个模块 介导 Ca2+/钙调蛋白的组成型一氧化氮合酶 将构建对后一种酶的控制。 利用这些 方法、底物特异性和催化的决定因素 将确定 CYP4A 酶的效率。