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，因为当被光激活时，它们会直接反应 并且不可逆地与DNA中的嘧啶碱基结合。光生物 这些呋喃香豆素的性质已被开发用于治疗 各种各样的疾病，尽管它们的发病机制 新陈代谢尚未得到很好的证实。投入了相当大的努力 记录呋喃香豆素在哺乳动物和鸟类中的代谢解毒作用 昆虫表明花椒毒素的主要代谢物，一种 线性呋喃香豆素，在昆虫和各种 脊椎动物物种，但在脊椎动物系统中，关于 与此相关的P450氨基酸决定因素(P450) 分解代谢。存在于一些食草性昆虫中的P450能够 使这些呋喃香豆素的线形和角形解毒，因此， 代表存在于可比的催化结构域的原型 脊椎动物的单加氧酶。克隆的细胞色素P6B1的特异性反应性 从凤尾蝶(黑燕尾)中分离出来的表明这些昆虫 幼虫至少表达两个呋喃香豆素代谢P45O：CYP6B1，它 主要代谢线性呋喃香豆素，以及另一种与CYP6B1相关的 P450，代谢角质呋喃香豆素。刻画人物形象 将依次进行细胞色素P6B1等位基因和细胞色素P6B1相关变异 确定呋喃香豆素代谢P45O的催化活性。 天然和突变的CYP6B1变异体将被用来识别氨基酸 它区分了线性导数和角导数。 目标是：1)充分定义底物的特性 使用一系列天然和人工合成的线性等位基因的研究 呋喃香豆素；2)克隆和表达编码另一种的cDNA 自然产生的同工酶，包括一种与CYP6B1相关的变异体 可能负责代谢角质呋喃香豆素，并定义 每个变种的底物特异性；3)预测和提炼 通过结构比较研究细胞色素P6B1的活性中心三级结构 与脊椎动物CYP2A5，它也代谢线性 呋喃香豆素黄毒素及其天然的CyP6B1变异体；4)至 通过诱变氨基酸来检验三级结构预测 可能在催化部位。这些特定的目标应该是分子上的 确定该昆虫P450中调节底物的关键氨基酸 对线形和角形呋喃香豆素的特异性，并提供 昆虫对呋喃香豆素抗性进化的研究进展 脊椎动物也会代谢这些剧毒化合物。

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.