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: 6179588
• 负责人: BETTIE SUE SILER MASTERS
• 金额: $ 22.36万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179588
• 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.

这项研究计划的重点是确定分子 CYP 4A基因亚家族的四个成员的催化特性 羟基化脂肪酸和类花生酸的细胞色素P450 主要地或专门地处于PSI位置。 这个组织的原始成员 密切相关（氨基酸水平同源性大于85%） P4504 A4亚家族，在本实验室同时分离 从怀孕兔子的肺和Kusunose的实验室， 炔雌醇处理的雄性家兔。 约翰逊等克隆并表达了 三种编码月桂酸β-羟化酶的肾脏cDNA， 图4A 5、4A 6和4A 7。 报道了一个编码CYP 4A 4的部分cDNA 通过Kusunose的实验室和完整的，全长cDNA分离 由约翰逊和马斯特斯的实验室联合开发。 虽然功能 这些细胞色素P450是未知的，大量的文献正在开发 其中它们通过控制血液动力学功能 血管张力 大脑和肾脏的微血管收缩20- 羟基二十碳四烯酸（ 花生四烯酸），浓度低于10 - 10 M。 由于 在肾脏中发现这些酶的高度序列同源性 和肺，它们在血流动力学控制中的意义，以及它们的程度， 底物特异性，计划实现以下具体目标：1） CYP 4A 7基因产物的表达将使用其他E. 大肠杆菌菌株以及其他载体，在存在和不存在 编码E.大肠杆菌伴侣蛋白（groEL和groES）。 2)的 这些细胞色素P450的重建将尝试使用 常规的超声处理的脂质制剂和通过掺入 脂质体（脂质囊泡）通过各种技术制备，包括 使用两亲性洗涤剂。3)定点突变， 从嵌合构建体中鉴定特异性决定簇将是 用每种CYP 4A酶（4A 4 - 4A 7）和突变体进行的试验将 用E.杆菌4)通过基于同源性的CYP 4A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Kim和Masters实验室获得，将被识别， 进行模块交换或定点诱变。 5)融合 含有CYP 4A亚家族成员之一和NADPH- 细胞色素P450还原酶和来自该酶的一个或多个模块。 介导Ca 2 +/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 将测定CYP 4A酶的效率。