CATALYTIC SITE IN FURANOCOUMARIN METABOLIC P450S

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，因为当它们被光活化时， 并且与DNA中的嘧啶碱基不可逆地结合。光生物学 这些呋喃香豆素的性质已被用于治疗 各种各样的疾病，即使他们的机制， 新陈代谢还没有很好地建立起来。投入了大量的努力， 记录了哺乳动物、鸟类、 和昆虫的研究表明，黄毒素的初级代谢产物， 线性呋喃香豆素，在昆虫和各种 脊椎动物物种，但鲜为人知的是，在脊椎动物系统中， 负责这些的P450氨基酸决定簇（P450） 分解代谢存在于一些食草昆虫中的P450能够 解毒这些呋喃香豆素的线性和角形形式， 代表存在于可比较的催化结构域中的原型 脊椎动物单加氧酶。克隆的CYP 6 B1的特异性反应性 黑燕尾蝶（Papilio polyxenes）表明，这些昆虫 幼虫表达至少两种呋喃香豆素代谢P450：CYP 6 B1， 主要代谢线性呋喃香豆素类，另一种与CYP 6 B1相关 P450，代谢角呋喃香豆素。表征 CYP 6 B1等位基因和CYP 6 B1相关变体将按顺序进行 以确定呋喃香豆素代谢P450中的催化位点反应性。 天然和突变型CYP 6 B1变体将用于鉴别氨基酸 其区分线性导数和角导数。 目的是：1）充分定义的底物特异性的 CYP 6 B1 v1/1v 2等位基因使用一系列天然和合成线性 2）克隆并表达编码呋喃香豆素的cDNA， 天然存在的同工酶，包括CYP 6 B1相关变体， 可能负责代谢角呋喃香豆素，并定义 每种变体的底物特异性; 3）预测和改进 CYP 6 B1活性位点三级结构的结构比较 与脊椎动物CYP 2A 5，它也代谢线性 呋喃香豆素黄毒素，并与天然CYP 6 B1变体; 4） 通过诱变氨基酸来测试三级结构预测 可能在催化位点。这些具体目标应该从分子上 确定这种昆虫P450中对调节底物至关重要的氨基酸 对线性和角型呋喃香豆素的特异性， 关于昆虫对呋喃香豆素抗药性的演变的信息， 以及脊椎动物，它们也代谢这些高毒性化合物。

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.