Schwarzer Dirk, Mootz Henning D, Linne Uwe, Marahiel Mohamed A
Biochemie, Fachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14083-8. doi: 10.1073/pnas.212382199. Epub 2002 Oct 16.
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 (TEII(srf)) and bacitracin (TEII(bac)). 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 TEII(srf), a K(M) of 0.9 microM 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 TEII(srf). 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.
非核糖体肽合成酶（NRPSs）利用氨基酸和羧酸等简单构件组装结构复杂的肽。由硫酯酶结构域催化的大环化或水解作用可实现产物释放，该硫酯酶结构域是NRPS酶的一个组成部分。先前通过基因敲除实验表明，由与NRPS基因簇相关的一个不同基因编码的II型硫酯酶（TEII）对于高效产物形成很重要。然而，TEIIs在非核糖体肽合成中的实际作用仍不清楚。在此，我们报道了两种此类TEII酶的生化特性，它们分别与肽抗生素表面活性素（TEII(srf)）和杆菌肽（TEII(bac)）的合成酶相关。这两种酶都能有效地再生附着在NRPSs的肽基载体蛋白（PCP）上的4'-磷酸泛酰巯基乙胺（4'PP）辅因子的错误酰化硫醇基团。对于TEII(srf)，测定其催化乙酰-PCP水解的米氏常数（K(M)）为0.9微摩尔，催化常数（k(cat)）为95分钟⁻¹。这两种酶还能水解非核糖体肽合成的中间体氨酰基或肽基PCP。然而，该反应不太可能具有生理相关性。在用TEII(srf)进行的研究中，脂肪酸合成的初级代谢的类似中间体如辅酶A衍生物和乙酰-酰基载体蛋白也未被显著水解。这些发现支持了这样一种模型，即TEIIs在非核糖体肽合成中的生理作用是再生错误酰化的NRPS，这是由于专用4'PP转移酶的混杂性导致NRPS酶从脱辅基形式向全酶形式转化，从而产生了错误酰化的NRPS，因为这些转移酶不仅使用游离辅酶A，还使用酰基辅酶A作为4'PP供体。
