Nonribosomal peptides (NRPs) are known sources of therapeutics. Some nonribosomal peptide synthetase assembly lines contain unique functional interrupted adenylation (A) domains, where nature has combined two different functional domains into one bifunctional enzyme. Most often these interrupted A domains contain a part of a methylation (M) domain embedded in their sequence. Herein, we aimed to emulate nature and create fully functional interrupted A domains by inserting two different noncognate M domains, KtzH(M_H) and TioS(M_3S), into a naturally occurring uninterrupted A domain, Ecm6(A₁T₁). We evaluated the engineered enzymes, Ecm6(A_1aM_HA_1bT₁) and Ecm6(A_1aM_3SA_1bT₁), by a series of radiometric assays and found that not only do they maintain A domain activity, but also they gain the site-specific methylation patterns observed in the parent M domain donors. These findings provide an exciting proof-of-concept for generating interrupted A domains as future tools to modify NRPs and increase the diversity and activity of potential therapeutics.

中文翻译：

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能够对氨基酸的侧链或核心进行腺苷酸化和选择性甲基化的工程双功能酶

非核糖体肽（NRPs）是治疗剂的已知来源。一些非核糖体肽合成酶装配线包含独特的功能性中断腺苷酸化（A）结构域，其中自然界将两个不同的功能性结构域组合成一个双功能酶。这些中断的A结构域最常见的是在其序列中嵌入一部分甲基化（M）结构域。在本文中，我们的目的是模拟性质和通过插入两个不同的非同源中号的域创建功能全面的中断A结构域，KtzH（M _ħ）和TIOS（M _3S），成天然存在的不间断的A结构域，Ecm6（A ₁ Ť ₁）。我们评估了工程化酶Ecm6（A _1a M _H A _1b T ₁）和Ecm6（A _1a M _3S A _1b T ₁），通过一系列放射分析发现，它们不仅保持A结构域活性，而且获得在亲本M结构域供体中观察到的位点特异性甲基化模式。这些发现为生成中断的A结构域提供了令人兴奋的概念证明，可作为未来修饰NRP并增加潜在疗法的多样性和活性的工具。