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Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-12-30 , DOI: 10.1021/acschembio.9b00929
Taylor A Lundy 1 , Shogo Mori 1 , Nishad Thamban Chandrika 1 , Sylvie Garneau-Tsodikova 1
Affiliation  

Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.

中文翻译:

可以催化三个反应的独特的中断腺苷酸化域的表征。

中断的腺苷酸化(A)结构域在其结构内包含辅助结构域,并且在非核糖体肽生物合成领域中受到越来越多的关注。它们已被证明具有有趣的功能和结构以及有希望的工程潜力。在这里,我们介绍了从哥伦布酰胺生物合成途径ColG(AMsMbA)前所未有的中断A域的特征。该中断的A结构域在A结构域的同一中断位点内包含两个背对背的甲基化(M)结构域,而以前,自然发生的报道和表征的中断的A结构域仅具有一个M结构域。通过一系列的放射和质谱分析,我们显示,在底物转移到载体硫醇化结构域ColG(T)后,第一个和第二个M结构域位点特异性甲基化了1-Ser的侧链氧和主链氮。这是首次报道的二甲基化背对背中断的A结构域的表征。通过这项工作获得的见识为未来位点特异性甲基化非核糖体肽的组合生物合成奠定了基础。
更新日期:2019-12-30
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