Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases
Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases
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DOI：
10.1073/pnas.212382199
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发表时间：
2002-10-29
影响因子：
11.1
通讯作者：
Marahiel, MA
中科院分区：
文献类型：
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作者：
Schwarzer, D;Mootz, HD;Marahiel, MA
非核糖体肽合成酶（NRPS）从简单的结构单元（如氨基酸和羧酸）组装结构复杂的肽。通过大环化或水解的产物释放由作为NRPS酶的整合部分的硫酯酶结构域催化。由与NRPS簇相关的不同基因编码的第二种II型硫酯酶（TEII）先前通过基因破坏的方式显示出对于有效的产物形成是重要的。然而，TEII在非核糖体肽合成中的实际作用仍然不清楚。在这里，我们报告了两个这样的TEII酶的生物化学表征，与肽抗生素表面活性素（TEIIIsrf）和杆菌肽（TEIIbac）的合成酶。两种酶均显示出有效地再生附接至NRPS的肽基载体蛋白（PCP）的4 '-磷酸泛酰巯基乙胺（4' PP）辅因子的错酰化巯基。对于TEIIsrf，测定乙酰-PCP水解的K-M为0.9 μ M，k（cat）为95 min（-1）。这两种酶也可以水解氨酰基或肽基PCP，非核糖体肽合成的中间体。然而，该反应不太可能具有生理相关性。初级代谢的类似中间体，如CoA衍生物和脂肪酸合成的乙酰-酰基载体蛋白，也没有显著水解，如TEIIisrf所研究的。这些发现支持了一种模型，其中TEII在非核糖体肽合成中的生理作用是错酰化NRPS的再生，这是由于专用4 'PP转移酶的混杂性导致NRPS酶的apo到holo转化，所述专用4' PP转移酶不仅使用游离CoA，而且使用酰基-CoA作为4 'PP供体。
Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEIIsrf) and bacitracin (TEIIbac). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEIIsrf, a K-M of 0.9 muM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEIIsrf. These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.