National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland Maynooth, Co. Kildare, Ireland.
Bioconjug Chem. 2009 Aug 19;20(8):1514-22. doi: 10.1021/bc900071j. Epub 2009 Jul 23.
No data exist on the ability of thiolation domains from fungal non-ribosomal peptide synthetases to undergo 4'-phosphopantetheinylation, using either biotinylated or fluorescently labeled coenzyme A analogues, mediated by 4'-phosphopantetheinyl transferases (PPTase). Yet, this is a key requirement to confirm the amino acid recognition function, and coding potential, of either non-ribosomal peptide synthetases or recombinantly expressed regions of these enzymes (e.g., didomains or modules). Moreover, determination of 4'-phosphopantetheinylation activity remains cumbersome. Here, we demonstrate that a recombinant fungal PPTase catalyzes the solution-phase transfer of either biotin- or fluorescein-labeled 4'-phosphopantetheine region of coenzyme A to a fungal thiolation domain, which is either part of a non-ribosomal peptide synthetase didomain (72 kDa), derived from Aspergillus fumigatus, or fused to a non-native protein (glutathione s-transferase). Significantly, we demonstrate that this reaction can unexpectedly occur when the target protein (4.4 pmol) is immobilized on a solid surface. These findings (i) confirm that thiolation domains of fungal origin, in native or non-native configuration, can accept modified 4'-phosphopantetheine residues via PPTase-mediated labeling and (ii) illustrate a novel, high-throughput method to determine PPTase activity.
尚无数据表明真菌非核糖体肽合酶的硫醇化结构域能够通过 4'-磷酸泛酰巯基乙胺基转移酶（PPTase）介导的生物素化或荧光标记辅酶 A 类似物发生 4'-磷酸泛酰巯基乙胺化，然而，这是确认非核糖体肽合酶或这些酶的重组表达区域（例如二结构域或模块）的氨基酸识别功能和编码潜力的关键要求。此外，4'-磷酸泛酰巯基乙胺化活性的测定仍然很繁琐。在这里，我们证明了一种重组真菌 PPTase能够在溶液相中催化生物素或荧光标记的辅酶 A 的 4'-磷酸泛酰巯基乙胺区域转移到真菌硫醇化结构域，该硫醇化结构域是来自烟曲霉的非核糖体肽合酶二结构域（72 kDa）的一部分，或融合到非天然蛋白质（谷胱甘肽 s-转移酶）上。重要的是，我们证明当目标蛋白（4.4 pmol）固定在固体表面上时，该反应可以出乎意料地发生。这些发现（i）证实了天然或非天然构型的真菌来源的硫醇化结构域可以通过 PPTase 介导的标记接受修饰的 4'-磷酸泛酰巯基乙胺残基，（ii）说明了一种新颖的、高通量测定 PPTase 活性的方法。
