Biosynthesis of clorobiocin: investigation of the transfer and methylation of the pyrrolyl-2-carboxyl moiety
Biosynthesis of clorobiocin: investigation of the transfer and methylation of the pyrrolyl-2-carboxyl moiety
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DOI：
10.1007/s00203-006-0190-9
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发表时间：
2007-03-01
影响因子：
2.8
通讯作者：
Heide, Lutz
中科院分区：
文献类型：
--
作者：
Anderle, Christine;Alt, Silke;Heide, Lutz
Clorobiocin 是一种氨基香豆素抗生素，含有 5-甲基吡咯基-2-羧基部分，通过酯键连接到脱氧糖上。该吡咯基部分对于抗生素与其生物靶标（旋转酶的 B 亚基）的结合非常重要。灭活实验表明，两种假定的酰基载体蛋白CloN5和CloN1以及两种假定的酰基转移酶CloN2和CloN7参与吡咯基-2-羧基部分向脱氧糖的转移。在本研究中，合成了吡咯基-2-羧基-N-乙酰半胱胺硫酯，并将其喂入cloN1(-)、cloN2(-)和cloN7(-)突变体，并通过HPLC和HPLC-MS分析了次级代谢产物的形成。在 cloN1(-) 和 cloN2(-) 突变体中观察到吡咯基-2-羧基部分的转移，但在 cloN7(-) 突变体中没有观察到，表明 CloN7 负责该反应。该转移的产物新氯霉素 109 没有进一步甲基化为 5-甲基吡咯基-2-羧基化合物，即氯霉素，表明酰基转移后不会发生甲基化。对突变体中 5-甲基吡咯基-2-羧酸的存在进行的其他研究以及甲基转移酶基因 cloN6 的灭活实验表明，CloN6 的甲基化和 CloN7 的酰基转移以协调的方式发生，需要两种蛋白质的存在才能有效形成产物。提出了氯霉素生物合成后期甲基化/酰基转移过程的机制，涉及 CloN1、CloN2、CloN5、CloN6 和 CloN7。
Clorobiocin is an aminocoumarin antibiotic containing a 5-methylpyrrolyl-2-carboxyl moiety, attached by an ester bond to a deoxysugar. This pyrrolyl moiety is important for the binding of the antibiotic to its biological target, the B subunit of gyrase. Inactivation experiments had shown that two putative acyl carrier proteins, CloN5 and CloN1, and two putative acyl transferases, CloN2 and CloN7, are involved in the transfer of the pyrrolyl-2-carboxyl moiety to the deoxysugar. In this study, pyrrolyl-2-carboxyl-N-acetylcysteamine thioester was synthesized and fed to cloN1(-), cloN2(-) and cloN7(-) mutants, and secondary metabolite formation was analyzed by HPLC and HPLC-MS. Transfer of the pyrrolyl-2-carboxyl moiety was observed in the cloN1(-) and cloN2(-) mutants, but not in the cloN7(-) mutant, suggesting that CloN7 is responsible for this reaction. The product of this transfer, novclobiocin 109, was not further methylated to the 5-methylpyrrolyl-2-carboxyl compound, i.e. clorobiocin, suggesting that methylation does not take place after the acyl transfer. Additional investigations for the presence of 5-methylpyrrolyl-2-carboxylic acid in the mutants, and inactivation experiments with the methyltransferase gene cloN6, suggested that methylation by CloN6 and acyl transfer by CloN7 take place in a concerted fashion, requiring the presence of both proteins for efficient product formation. A mechanism for the methylation/acyl transfer process in the late steps of clorobiocin biosynthesis, involving CloN1, CloN2, CloN5, CloN6 and CloN7 is suggested.