CATALYTIC SITE IN FURANOCOUMARIN METABOLIC P450S

呋喃香豆素代谢 P450S 中的催化位点

基本信息

批准号：
2022768
负责人：
Mary A Schuler
金额：
$ 23.48万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-12-01 至 1998-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2022768
关键词：
RNase protection assay active sites alleles chemical structure function cytochrome P450 cytotoxicity detoxification enzyme structure enzyme substrate furocoumarin isozymes molecular cloning northern blottings physical model polymerase chain reaction protein sequence radionuclides tissue /cell culture

项目摘要

Furanocoumarins are naturally occurring secondary plant metabolites which are extremely phototoxic because when photoactivated they react directly and irreversibly with pyrimidine bases in DNA. The photobiologic properties of these furanocoumarins have been exploited for the treatment of a wide variety of diseases even though the mechanisms for their metabolism have not been well established. Considerable effort invested in documenting metabolic detoxification of furanocoumarins in mammals, birds and insects has suggested that the primary metabolites for xanthotoxin, a linear furanocoumarin, are identical in insects and a variety of vertebrate species and yet little is known, in vertebrate systems, about the P450 amino acid determinants (P450s) responsible for these catabolisms. P450s which exist in some herbivorous insects are capable of detoxifying linear and angular forms of these furanocoumarins and thus, represent prototypes for the catalytic domains existing in comparable vertebrate monooxygenases. The specific reactivities for CYP6B1 cloned from Papilio polyxenes (black swallowtail) indicate that these insect larvae express at least two furanocoumarin-metabolic P45Os: CYP6B1, which principally metabolizes linear furanocoumarins, and another CYP6B1-related P450, which metabolizes angular furanocoumarins. Characterization of the CYP6B1 alleles and the CYP6B1-related variant will be undertaken in order to define catalytic site reactivities in furanocoumarin-metabolic P45Os. Natural and mutant CYP6B1 variants will be used to identify amino acids which discriminate between linear and angular derivatives. The objectives are: 1) To fully define the substrate specificities of the CYP6B1v1/1v2 alleles using a series of natural and synthetic linear furanocoumarins; 2) To clone and express cDNAs encoding the other naturally occurring isozymes, including a CYP6B1-related variant which maybe responsible for metabolizing angular furanocoumarins, and to define the substrate specificities for each variant; 3) To predict and refine the active site tertiary structure for CYP6B1 by comparison of its structure with the vertebrate CYP2A5, which also metabolizes the linear furanocoumarin xanthotoxin, and with the natural CYP6B1 variants; 4) To test the tertiary structure predictions by mutagenizing amino acids potentially in the catalytic site. These specific aims should molecularly define amino acids in this insect P450 critical for modulating substrate specificity towards linear and angular furanocoumarins and provide information on the evolution of furanocoumarin resistances in insects as well as vertebrates which also metabolize these highly toxic compounds.

呋喃香豆素是天然存在的植物次生代谢物，具有极强的光毒性，因为当光激活时它们会直接发生反应并与DNA中的嘧啶碱基不可逆地结合。光生物学这些呋喃香豆素的特性已被用于治疗多种疾病的发生，尽管其发病机制新陈代谢尚未得到很好的建立。投入了大量的精力记录哺乳动物、鸟类中呋喃香豆素的代谢解毒和昆虫表明，黄蜂毒素的主要代谢物是线性呋喃香豆素，在昆虫和多种昆虫中是相同的脊椎动物物种，但在脊椎动物系统中，我们知之甚少负责这些的 P450 氨基酸决定簇 (P450s) 分解代谢。存在于一些草食性昆虫中的 P450 能够对这些呋喃香豆素的线性和角形形式进行解毒，因此，代表类似中存在的催化域的原型脊椎动物单加氧酶。克隆的 CYP6B1 的特异性反应来自 Papilio Polyxenes（黑色燕尾）的昆虫表明这些昆虫幼虫表达至少两种呋喃香豆素代谢 P45O：CYP6B1，主要代谢线性呋喃香豆素，以及另一种 CYP6B1 相关的 P450，代谢角呋喃香豆素。的表征 CYP6B1等位基因和CYP6B1相关变体将按顺序进行定义呋喃香豆素代谢 P45O 中的催化位点反应性。天然和突变 CYP6B1 变体将用于鉴定氨基酸区分线性导数和角度导数。目标是： 1) 充分定义底物的特异性 CYP6B1v1/1v2等位基因使用一系列天然和合成的线性呋喃香豆素类； 2) 克隆并表达编码另一种的cDNA 天然存在的同工酶，包括 CYP6B1 相关变体可能负责代谢角呋喃香豆素，并定义每个变体的底物特异性； 3) 预测和完善 CYP6B1 活性位点三级结构的结构比较与脊椎动物 CYP2A5 一起，它也代谢线性呋喃香豆素黄毒素，以及天然 CYP6B1 变体； 4) 至通过氨基酸诱变测试三级结构预测可能在催化位点。这些具体目标应该从分子角度定义该昆虫 P450 中对调节底物至关重要的氨基酸对线性和有角呋喃香豆素的特异性，并提供有关昆虫呋喃香豆素抗性进化的信息以及也代谢这些剧毒化合物的脊椎动物。