Design, Synthesis, and Biophysical Evaluation of Mechanism-Based Probes for Condensation Domains of Nonribosomal Peptide Synthetases.,ACS Chemical Biology

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Design, Synthesis, and Biophysical Evaluation of Mechanism-Based Probes for Condensation Domains of Nonribosomal Peptide Synthetases.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-06-22 , DOI: 10.1021/acschembio.0c00411
Ce Shi ₁ , Bradley R Miller ₂ , Evan M Alexander ₁ , Andrew M Gulick ₂ , Courtney C Aldrich ₁

Affiliation

Nonribosomal peptide synthetases (NRPSs) are remarkable modular enzymes that synthesize peptide natural products. The condensation (C) domain catalyzes the key amide bond-forming reaction, but structural characterization with bound donor and acceptor substrates has proven elusive. We describe the chemoenzymatic synthesis of condensation domain probes C1 and C2 designed to cross-link the donor and acceptor substrates within the condensation domain active site. These pantetheine probes contain nonhydrolyzable ketone and α,α-difluoroketone isosteres of the native thioester linkage. Using the bimodular NRPS responsible for synthesis of the siderophore enterobactin as a model system, probe C2 was shown by surface plasmon resonance (SPR) to stabilize an intermolecular interaction between the peptidyl carrier protein (PCP) and C domains in EntB and EntF, respectively, with a dissociation constant of 1–2 nM, whereas the unmodified holo-EntB showed no interaction with EntF. The described condensation domain chemical probes provide powerful tools to study dynamic multifunctional NRPS systems.

中文翻译：

非核糖体肽合成酶缩合域的基于机理的探针的设计，合成和生物物理评估。

非核糖体肽合成酶（NRPS）是合成肽天然产物的出色模块化酶。缩合（C）域催化关键的酰胺键形成反应，但是结合供体和受体底物的结构表征已被证明难以捉摸。我们描述了缩合结构域探针C1和C2的化学酶法合成，设计用于交联在缩合结构域活性位点内的供体和受体底物。这些泛肽探针包含天然硫酯键的不可水解的酮和α，α-二氟酮的等排体。使用负责合成铁载体肠杆菌素的双模块NRPS作为模型系统，探针C2表面等离振子共振（SPR）可以稳定EntB和EntF中肽基载体蛋白（PCP）和C结构域之间的分子间相互作用，解离常数为1-2 nM，而未修饰的holo -EntB则没有与EntF交互。所述缩合域化学探针提供了强大的工具来研究动态多功能NRPS系统。

更新日期：2020-07-17

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