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: 2734463
• 负责人: BETTIE SUE SILER MASTERS
• 金额: $ 21.37万
• 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/2734463
• 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.

本研究方案的重点是分子的测定。 细胞色素P4A基因亚家族四个成员的催化性质 使脂肪酸和二十烷类化合物羟化的细胞色素P450 主要或完全处于PSI位置。这个组织的原始成员 密切相关(在氨基酸水平上同源性超过85%) 亚家族为P4504A4，在本实验室同时分离 从怀孕兔子的肺中提取，从Kusunose的实验室提取 用黄体酮处理的雄兔。Johnson等人克隆和表达 三个肾脏编码月桂酸psi-羟基酶的cDNA现在被称为 CYP 4A5、4A6和4A7。报道了一个编码细胞色素P4A4的部分基因 通过Kusunose的实验室，获得了完整的、全长的cDNA 由约翰逊的实验室和硕士的实验室联合。虽然这些函数 其中细胞色素P450尚不为人所知，大量文献正在开发中 在这种情况下，它们通过控制血流动力学功能 血管张力。大脑和肾脏的微血管收缩了20- 羟基二十碳四烯酸(psi-羟基化产物 花生四烯酸)，浓度小于10-10M。由于 在肾脏中发现的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 底物专一性，计划实现以下具体目标：1) 我们将利用其他E. 在存在和不存在 编码大肠杆菌伴侣蛋白(GroEL和GroES)的质粒。2) 这些细胞色素P450的重组将尝试使用 传统的超声脂质制剂，并通过将其加入到 通过各种技术制备的脂质体(脂泡)，包括 使用两亲性洗涤剂。3)基于基因的定点突变 从嵌合结构中识别特异性决定因素将是 用每一种CYP4A酶(4A4-4A7)执行，突变株将 在大肠杆菌中表达。4)通过对细胞色素P4A的同源性建模 利用巨大芽孢杆菌血红素结构域的酶， 可能参与对接的这些酶的模块和区域 NADPH-细胞色素P450还原酶，其结构已被 由金实验室和马斯特斯实验室获得的，将被识别并 进行模块交换或定点突变。5)融合 含有CYP4A亚家族成员之一和NADPH-的蛋白质 细胞色素P450还原酶和一个或多个来自 介导钙/钙调素的结构性一氧化氮合酶 将构建对后一种酶的控制。利用这些 方法、底物特异性的决定因素和催化 将测定CYP4A酶的效率。