Freitag Anja, Wemakor Emmanuel, Li Shu-Ming, Heide Lutz
Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
Chembiochem. 2005 Dec;6(12):2316-25. doi: 10.1002/cbic.200500252.
Clorobiocin is an aminocoumarin antibiotic containing a pyrrole-2-carboxyl moiety, attached through an ester bond to a deoxysugar. The pyrrole moiety is important for the binding of the antibiotic to its biological target, gyrase. The complete biosynthetic gene cluster for clorobiocin has been cloned and sequenced from the natural producer, Streptomyces roseochromogenes DS 12.976. In this study, the genes cloN1 and cloN7 were deleted separately from a cosmid containing the complete clorobiocin cluster. The modified cosmids were introduced into the genome of the heterologous host Streptomyces coelicolor M512 by using the integration functions of the PhiC31 phage. While a heterologous producer strain harbouring the intact clorobiocin biosynthetic gene cluster accumulated clorobiocin, the cloN1- and cloN7-defective integration mutants accumulated a clorobiocin derivative that lacked the pyrrole-2-carboxyl moiety, while also producing free pyrrole-2-carboxylic acid. The structures of these metabolites were confirmed by NMR and MS analysis. These results showed that CloN1 and CloN7, together with the previously investigated CloN2, are involved in the transfer of the pyrrole-2-carboxyl moiety to the deoxysugar of clorobiocin. A possible mechanism for the role of these three proteins in the acyl-transfer process is suggested.
氯新生霉素是一种氨基香豆素类抗生素，含有一个吡咯 -2- 羧基部分，通过酯键与一种脱氧糖相连。吡咯部分对于抗生素与其生物靶点——回旋酶的结合很重要。氯新生霉素完整的生物合成基因簇已从天然产生菌玫瑰色链霉菌DS 12.976中克隆并测序。在本研究中，基因cloN1和cloN7分别从含有完整氯新生霉素基因簇的粘粒中缺失。利用PhiC31噬菌体的整合功能，将修饰后的粘粒导入异源宿主天蓝色链霉菌M512的基因组中。携带完整氯新生霉素生物合成基因簇的异源产生菌株积累了氯新生霉素，而cloN1和cloN7缺陷型整合突变体积累了一种缺少吡咯 -2- 羧基部分的氯新生霉素衍生物，同时还产生了游离的吡咯 -2- 羧酸。这些代谢产物的结构通过核磁共振（NMR）和质谱（MS）分析得以确认。这些结果表明，CloN1和CloN7与之前研究的CloN2一起，参与了吡咯 -2- 羧基部分向氯新生霉素脱氧糖的转移。文中提出了这三种蛋白质在酰基转移过程中作用的一种可能机制。
