Laboratory of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka, 577-8502, Japan.
Chembiochem. 2020 Nov 2;21(21):3056-3061. doi: 10.1002/cbic.202000206. Epub 2020 Jul 17.
An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.
天然产物生物合成中的一个重要挑战是人工肽的生物合成设计和生产。最有前途的策略之一是重新编程腺苷酸化（A）结构域，以扩大非核糖体肽合酶（NRPS）的底物 repertoire。因此，精确检测 A 结构域底物结合口袋中的细微结构变化可能会加速其重新编程。在这里，我们展示了一种酶联免疫吸附测定（ELISA），使用小分子探针的组合可以检测 A 结构域中底物结合口袋残基取代的影响。当与一组芳基酸 A 结构域变体（总共 9 个变体）结合使用时，ELISA 可以分析它们活性位点结构的细微差异。此外，基于 ELISA 的筛选能够从原始的 45 个底物池中识别出具有接受非天然底物的底物结合口袋的变体。这些研究表明，ELISA 是一种可靠的平台，可以深入了解 A 结构域的活性位点特性，并可应用于 NRPS A 结构域的重新编程。
