A Capture Strategy for the Identification of Thio-Templated Metabolites
A Capture Strategy for the Identification of Thio-Templated Metabolites
复制标题
识别硫代模板代谢物的捕获策略
DOI：
10.1021/acschembio.1c00437
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发表时间：
2021
影响因子：
4
通讯作者：
Watanabe, Coran M.
中科院分区：
文献类型：
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作者：
Washburn, Lauren A.;Nepal, Keshav K.;Watanabe, Coran M.
非核糖体肽合成酶和聚酮合成酶系统是复杂酶学的所在地，并产生具有重要治疗价值的化合物。尽管如此，由于它们的底物仍然通过硫酯键与酶结合，因此它们仍然难以表征。在这里，我们已经开发出一种策略，直接捕获和表征的硫酯结合的酶的中间体，并应用该策略的azinomycin的生物合成途径。该方法最初应用于体外评估其功效，随后转移到原位杂交，其中感兴趣的蛋白质从天然生物体中分离出来，以避免需要提供底物。当非核糖体肽合成酶AziA3从Sahachiroi链霉菌中分离出来时，捕获策略揭示了AziA3在azinomycins的环氧化物部分形成的后期阶段中的功能。该策略进一步验证了在体外与参与大肠杆菌素生物合成的非核糖体肽合成酶。从长远来看，这种方法将被用来表征硫酯结合的代谢物不仅在azinomycin生物合成途径，但也其他隐蔽代谢途径。
Nonribosomal peptide synthetase and polyketide synthase systems are home to complex enzymology and produce compounds of great therapeutic value. Despite this, they have continued to be difficult to characterize due to their substrates remaining enzyme-bound by a thioester bond. Here, we have developed a strategy to directly trap and characterize the thioester-bound enzyme intermediates and applied the strategy to the azinomycin biosynthetic pathway. The approach was initially appliedin vitroto evaluate its efficacy and subsequently moved to anin situsystem, where a protein of interest was isolated from the native organism to avoid needing to supply substrates. When the nonribosomal peptide synthetase AziA3 was isolated fromStreptomyces sahachiroi, the capture strategy revealed AziA3 functions in the late stages of epoxide moiety formation of the azinomycins. The strategy was further validatedin vitrowith a nonribosomal peptide synthetase involved in colibactin biosynthesis. In the long term, this method will be utilized to characterize thioester-bound metabolites within not only the azinomycin biosynthetic pathway but also other cryptic metabolite pathways.