Lima Stella T, Pasquale Michael A, Noyon M Rafiul O K, Clark Elizabeth A, Laws Corinne R, Hematian Shabnam, Chekan Jonathan R
Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, North Carolina 27402, United States.
J Am Chem Soc. 2025 Jun 18;147(24):20284-20293. doi: 10.1021/jacs.4c15470. Epub 2025 Jun 5.
Side chain cross-linking of amino acids is a challenging oxidative enzymatic reaction that has largely been confined to heme and iron sulfur cluster containing enzymes. However, the recent discovery of plant BURP-domain peptide cyclases (BpCs) responsible for the biosynthesis of burpitide natural products has demonstrated that copper-dependent enzymes are able to generate similar cross-links. For example, ArbB2 is responsible for the formation of a Tyr-phenol-O to Leu-C bond observed in arabipeptin A, a cyclopeptide alkaloid isolated from the well-known plant . To investigate this intriguing enzyme family in more detail, we developed minimal peptide substrates for ArbB2, which enabled quantitative studies. By examining reductant dependence, we achieved catalytic turnover for the first time in this enzyme family, which allowed for kinetic, mutational, substrate scope, and multicore processing analyses. Additionally, we established the dioxygen dependence and confirmed the absence of hydrogen peroxide as a side product in the catalytic system. Finally, we extended our study to other BpCs involved in cyclopeptide alkaloid biosynthesis, demonstrating that our findings apply across additional members of this enzyme family. Ultimately, this work provides fundamental insights into a new, widespread family of copper-dependent peptide cyclases and lays the groundwork for future mechanistic investigations.
氨基酸的侧链交联是一种具有挑战性的氧化酶促反应，在很大程度上仅限于含血红素和铁硫簇的酶。然而，最近发现负责burpitide天然产物生物合成的植物BURP结构域肽环化酶（BpCs）表明，铜依赖性酶能够产生类似的交联。例如，ArbB2负责在阿拉伯肽A中观察到的从酪氨酸酚氧到亮氨酸碳的键的形成，阿拉伯肽A是一种从著名植物中分离出的环肽生物碱。为了更详细地研究这个有趣的酶家族，我们为ArbB2开发了最小肽底物，从而能够进行定量研究。通过研究对还原剂的依赖性，我们首次在这个酶家族中实现了催化周转，这使得我们能够进行动力学、突变、底物范围和多核加工分析。此外，我们确定了对双氧的依赖性，并证实催化体系中没有过氧化氢作为副产物。最后，我们将研究扩展到参与环肽生物碱生物合成的其他BpCs，表明我们的发现适用于这个酶家族的其他成员。最终，这项工作为一个新的、广泛存在的铜依赖性肽环化酶家族提供了基本见解，并为未来的机制研究奠定了基础。
