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Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity
Biochemistry ( IF 2.9 ) Pub Date : 2018-03-13 00:00:00 , DOI: 10.1021/acs.biochem.8b00076
Kenneth D. Clevenger 1 , Rosa Ye 2 , Jin Woo Bok 3 , Paul M. Thomas 1, 4 , Md Nurul Islam 2 , Galen P. Miley 5 , Matthew T. Robey 4 , Cynthia Chen 2 , KaHoua Yang 3 , Michael Swyers 2 , Edward Wu 2 , Peng Gao 1 , Chengcang C. Wu 2 , Nancy P. Keller 3 , Neil L. Kelleher 1, 4, 5
Affiliation  

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-CT and the internal C-domains as BenZ-C1 and BenZ-C2. Unexpectedly, we also uncovered evidence suggesting BenY-CT or a yet to be identified protein mediates benzodiazepine formation, representing the first reported benzodiazepine synthase enzymatic activity. This work informs understanding of what defines a fungal CT domain and shows how the FAC-MS platform can be used as a tool for in vivo analyses of specialized metabolite biosynthesis and for the discovery and dissection of new enzyme activities.

中文翻译:

使用真菌人工染色体与代谢组学评分(FAC-MS)审问苯并malvin生物合成:苯二氮卓合酶活性的发现。

苯二氮卓类苯并malvin A / D是真菌衍生的专门代谢物和P受体NK1的抑制剂,由三基因非核糖体肽合成酶簇生物合成。在这里,我们利用具有代谢组学评分(FAC-MS)的真菌人工染色体来进行分子遗传途径解剖和靶向代谢组学分析,以指定苯并丙氨酸生物合成途径中每个域的体内作用。FAC-MS的使用将末端环化缩合结构域确定为BenY-C T,将内部C结构域确定为BenZ-C 1和BenZ-C 2。出乎意料的是,我们还发现了暗示BenY-C T的证据或尚未确定的蛋白质介导苯并二氮杂formation的形成,代表了首次报道的苯并二氮杂合酶的酶促活性。这项工作有助于了解定义真菌C T结构域的内容,并显示FAC-MS平台如何用作体内专门代谢产物生物合成的分析工具以及新酶活性的发现和分解工具。
更新日期:2018-03-13
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