Larbat Romain, Hehn Alain, Hans Joachim, Schneider Sarah, Jugdé Hélène, Schneider Bernd, Matern Ulrich, Bourgaud Frédéric
UMR 1121 Nancy Université-Institut National de la Recherche Agronomique Agronomie Environnement Nancy-Colmar, 2 Avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy, France.
J Biol Chem. 2009 Feb 20;284(8):4776-85. doi: 10.1074/jbc.M807351200. Epub 2008 Dec 19.
The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Using cDNA probes inferred from CYP71AJ1, three orthologs were isolated from Apium graveolens (CYP71AJ2) and Pastinaca sativa (CYP71AJ3 and -4) and functionally expressed in yeast cells. CYP71AJ2 and CYP71AJ3 displayed psoralen synthase activity, whereas CYP71AJ4 only catalyzed the conversion of (+)-columbianetin to angelicin and negligible amounts of a hydroxylated columbianetin by-product. CYP71AJ4 thus constitutes the first fully characterized P450 monooxygenase specific for the angular furanocoumarin pathway. The angelicin synthase exhibited an apparent K(m) of 2.1 +/- 0.4 microm for (+)-columbianetin and a k(cat) of 112 +/- 14 min(-1). Moreover, the use of 3'-deuterated (+)-columbianetin as substrate led to an almost complete "metabolic switch," resulting in the synthesis of anti-3'-hydroxy-3'-deuterated(+)-columbianetin. This confirms that angelicin synthase attacks columbianetin by syn-elimination of hydrogen from C-3'. Sequence comparison between psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity, whereas the identity dropped to 40% in those regions thought to provide the substrate recognition sites. Accordingly, CYP71AJ3 and CYP71AJ4 might be derived from a common ancestor of unknown functionality by gene duplication and subsequent molecular evolution.
在分子水平上，人们对线性和角型呋喃香豆素的生物合成仍知之甚少，目前仅从药用阿米芹中鉴定出补骨脂素合酶（CYP71AJ1）。利用从CYP71AJ1推断出的cDNA探针，从芹菜（CYP71AJ2）和欧洲防风草（CYP71AJ3和-4）中分离出三个直系同源基因，并在酵母细胞中进行了功能表达。CYP并在酵母细胞中进行了功能表达。CYP71AJ2和CYP71AJ3表现出补骨脂素合酶活性，而CYP71AJ4仅催化（+）-哥伦比亚内酯转化为当归素以及极少量的羟基化哥伦比亚内酯副产物。因此，CYP71AJ4构成了第一个完全表征的、对角型呋喃香豆素途径具有特异性的P450单加氧酶。当归素合酶对（+）-哥伦比亚内酯的表观K（m）为2.1±0.4微摩尔，k（cat）为112±14分钟-1。此外，使用3'-氘代（+）-哥伦比亚内酯作为底物导致几乎完全的“代谢转换”，从而合成了反式-3'-羟基-3'-氘代（+）-哥伦比亚内酯。这证实了当归素合酶通过从C-3'顺式消除氢来攻击哥伦比亚内酯。补骨脂素合酶（CYP71AJ3）和当归素合酶（CYP71AJ4）之间的序列比较显示同一性为70%，而在那些被认为提供底物识别位点的区域，同一性降至40%。因此，CYP71AJ3和CYP71AJ4可能是通过基因复制和随后的分子进化，从功能未知的共同祖先衍生而来。
