NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein.
NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein.
复制标题
NADPH-细胞色素 P-450 氧化还原酶基因组织与蛋白质的结构域相关。
DOI：
10.1021/bi00494a009
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Kasper,CB
中科院分区：
文献类型：
--
作者：
Porter,TD;Beck,TW;Kasper,CB
托德D. Porter，§托马斯W.贝克，11岁和查尔斯B。Kasper * McArdle癌症研究实验室，威斯康星大学-麦迪逊分校，麦迪逊，威斯康星州53706接收于1990年2月12日;修订的Mandarpt接收于1990年6月20日摘要：使用鼠肝NADPH-细胞色素P-450氧化还原酶的cDNA克隆从Wistar-Furth近交系大鼠基因组DNA文库中分离基因组克隆。鉴定了包含P-450还原酶基因的编码区和3 '非翻译片段的15个外显子，跨越3个X-Charon 35克隆中包含的20个DNA酶。这个单拷贝基因的结构揭示了外显子和蛋白质结构域之间的一般对应关系，其中负责将还原酶锚定在微粒体膜上的片段和参与FMN、FAD和NADPH结合的几个片段由离散的外显子编码。jADH-细胞色素P-450氧化还原酶是一种78 225-道尔顿的黄素蛋白，与大多数真核细胞的内质网相关（威廉姆斯和Kamin，1962;菲利普斯和Langdon，1962）。该蛋白质负责从NADPH到细胞色素P-450的电子转移（Lu等人，1969），并且其不寻常之处在于其含有各1摩尔的FAD和FMN（Iyanagi & Mason，1973）。我们先前报道了大鼠肝P-450还原酶的氨基酸序列，其是从重叠克隆的cDNA中确定的（Porter & Kasper，1985），并报道了基于计算机辅助比较氨基酸序列与其他几种确定结构的黄素蛋白的酶的结构表征（Porter & Kasper，1986）。残基77 - 228与细菌黄素蛋白同源，表明还原酶的这一部分可能结合FMN;利用该区域中几个残基的定点诱变的研究为这一假设提供了强有力的支持（Shen et al.，1989年）。羧基端的几个片段与两个含FAD的蛋白质铁氧还蛋白-NADP+还原酶和NADH-细胞色素b5还原酶具有相当大的相似性，表明该还原酶的这一部分与FAD结合。比较这些蛋白质的谷胱甘肽还原酶，黄素蛋白的三维结构是已知的，允许暂定识别FAD和辅因子结合片段在这些蛋白质。P-450还原酶和这些FAD结合蛋白之间的相似性，加上与细菌flavodoxins的氨基末端结构域的同源性，表明还原酶通过这两组flavoproteins的祖先基因的融合而产生。为了进一步验证这一假设，我们克隆了大鼠P-450还原酶基因并对其进行了鉴定。外显子组织的分析揭示了外显子和拟议的功能或结构域之间的一般对应关系，并支持了P-450还原酶从两个不同的黄素蛋白基因的融合进化而来的假设。实验步骤基因组DNA文库的构建和筛选。从雄性Wistar-Furth大鼠肝脏中分离细胞核。这项工作得到了美国国立卫生研究院的赠款CA-22484、CA-09020和CA-09135的支持。本文中的核酸序列已在GenBank中登录，登录号为J05291。8现住址：密歇根大学医学院生物化学系，安阿伯，MI 48109。11现地址：国家癌症研究所，病毒致癌实验室，弗雷德里克癌症研究机构，弗雷德里克，MD 21701。
Todd D. Porter, § Thomas W. Beck, 11 and Charles B. Kasper* McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, Wisconsin 53706 Received February 12, 1990; Revised Manuscript Received June 20, 1990 abstract: cDNA clones to ratliver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 X-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.^ jADPH-cytochrome P-450 oxidoreductase is a 78 225-dalton flavoprotein associated with the endoplasmic reticulum of most eukaryotic cells (Williams & Kamin, 1962; Phillips & Langdon, 1962). The protein is responsible for electron transfer from NADPH to the cytochromes P-450 (Lu et al., 1969) and is unusual in that it contains 1 mol each of FAD and FMN (Iyanagi & Mason, 1973). We previously reported the amino acid sequence of rat liver P-450 reductase as de-termined from overlapping cloned cDNAs (Porter & Kasper, 1985) and have reported a structural characterization of the enzyme based on a computer-assisted comparison of the amino acid sequence to several other flavoproteins of defined structure (Porter & Kasper, 1986). Residues 77-228 are homologous with the bacterial flavodoxins, indicating that this portion of the reductase is likely to bind FMN; studies utilizing site-directed mutagenesis of several residues in this region have provided strong support for this hypothesis (Shen et al., 1989). Several segments in the carboxy-terminal regionshow con-siderable similarity to two FAD-containing proteins, ferre-doxin-NADP+ reductase and NADH-cytochrome b5 reduc-tase, suggesting that this portion of the reductase binds FAD. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has allowed tentative identification of FAD-and cofactor-binding segments in these proteins. The similarity between P-450 reductase and these FAD-binding proteins, coupled with the homology of the amino-terminal domain with the bacterial flavodoxins, suggests that thereductase arose through a fusion of the ancestral genes for these two sets of flavoproteins. To further evaluate this hypothesis, we have cloned and charac-terized the rat P-450 reductase gene. Analysis of the exon organization reveals a general correspondence between exons and proposed functional or structural domains, and supports the postulatethat P-450reductase evolved from a fusion of two different flavoprotein genes. Experimental Procedures Construction and Screening of the Genomic DNA Library. Nuclei were isolated from male Wistar-Furth rat liver fThis work was supported by Grants CA-22484, CA-09020, and CA-09135 from the National Institutes of Health.* The nucleic acidsequence in this paper has been submitted to Gen-Bank under Accession Number J05291. 8 Present address: Department of Biological Chemistry, Medical School, The University of Michigan, Ann Arbor, MI 48109. 11 Present address: National Cancer Institute, Laboratory for Viral Carcinogenesis, Frederick Cancer Research Facility, Frederick, MD 21701.