Rubin Garret M, Patel Krishna P, Jiang Yujia, Ishee Alivia C, Seabra Gustavo, Bruner Steven D, Ding Yousong
Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development, University of Florida, Gainesville, Florida 31610, United States.
Department of Chemistry, University of Florida, Gainesville, Florida 31611, United States.
ACS Chem Biol. 2024 Dec 20;19(12):2525-2534. doi: 10.1021/acschembio.4c00626. Epub 2024 Dec 4.
Graspetides are a diverse family of ribosomally synthesized and post-translationally modified peptides with unique macrocyclic structures formed by ATP-grasp enzymes. Group 11 graspetides, including prunipeptin, feature both macrolactone and macrolactam cross-links. Despite the known involvement of a single ATP-grasp cyclase in the dual macrocyclizations of groups 5, 7, and 11 graspetides, detailed mechanistic insights into these enzymes remain limited. Here, we reconstructed prunipeptin biosynthesis from using recombinant PruA and PruB macrocyclase. PruB exhibited kinetic behavior similar to other characterized graspetide cyclases, with a notably higher , likely due to utilization of an ATP-regeneration system. The X-ray crystal structure of PruB revealed distinct features as compared to groups 1 and 2 enzymes. Site-directed mutagenesis identified critical roles of key residues for the PruB reaction, including the DxR motif conserved in other graspetide cyclases. Additionally, computational modeling of the PruA/PruB cocomplex uncovered substrate interactions and suggested that PruB first catalyzes a macrolactone bond formation on PruA. This study enhances our understanding of ATP-grasp enzyme mechanisms in graspetide biosynthesis and provides insights for engineering these enzymes for future applications.
抓握肽是一类通过核糖体合成并经翻译后修饰的肽，具有由ATP抓握酶形成的独特大环结构。第11组抓握肽，包括李属肽，具有大环内酯和大环内酰胺交联结构。尽管已知单个ATP抓握环化酶参与第5、7和11组抓握肽的双重大环化过程，但对这些酶的详细机制了解仍然有限。在此，我们使用重组PruA和PruB大环化酶从[具体来源未给出]重建了李属肽的生物合成。PruB表现出与其他已表征的抓握肽环化酶相似的动力学行为，其[具体参数未给出]显著更高，这可能是由于利用了ATP再生系统。与第1组和第2组酶相比，PruB的X射线晶体结构显示出不同的特征。定点诱变确定了关键残基对PruB反应的关键作用，包括在其他抓握肽环化酶中保守的DxR基序。此外，PruA/PruB共复合物的计算模型揭示了底物相互作用，并表明PruB首先催化PruA上的大环内酯键形成。这项研究增进了我们对抓握肽生物合成中ATP抓握酶机制的理解，并为这些酶的工程改造以用于未来应用提供了见解。
