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A Capture Strategy for the Identification of Thio-Templated Metabolites
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2021-08-23 , DOI: 10.1021/acschembio.1c00437
Lauren A Washburn 1 , Keshav K Nepal 1 , Coran M H Watanabe 1
Affiliation  

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 applied in vitro to evaluate its efficacy and subsequently moved to an in situ system, 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 from Streptomyces sahachiroi, the capture strategy revealed AziA3 functions in the late stages of epoxide moiety formation of the azinomycins. The strategy was further validated in vitro with 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.

中文翻译:

一种用于鉴定硫代模板化代谢物的捕获策略

非核糖体肽合成酶和聚酮化合物合成酶系统是复杂酶学的发源地,可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,我们开发了一种直接捕获和表征硫酯结合酶中间体的策略,并将该策略应用于阿奇霉素生物合成途径。该方法最初在体外应用以评估其功效,随后转移到原位系统,在该系统中从天然生物体中分离出感兴趣的蛋白质以避免需要提供底物。当非核糖体肽合成酶 AziA3 从Streptomyces sahachiroi中分离出来,捕获策略揭示了 AziA3 在阿齐霉素环氧化物部分形成后期的功能。该策略在体外用参与大肠杆菌素生物合成的非核糖体肽合成酶进一步验证。从长远来看,这种方法将用于表征硫酯结合的代谢物,不仅在阿齐霉素生物合成途径中,而且在其他隐秘代谢物途径中。
更新日期:2021-09-17
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