In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe.
In Vitro Characterization of the Colibactin-Activating Peptidase ClbP Enables Development of a Fluorogenic Activity Probe.
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大肠杆菌素激活肽酶 ClbP 的体外表征有助于开发荧光活性探针。
DOI：
10.1021/acschembio.9b00069
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发表时间：
2019
影响因子：
4
通讯作者：
Balskus,EmilyP
中科院分区：
文献类型：
--
作者：
Volpe,MatthewR;Wilson,MatthewR;Brotherton,CarolynA;Winter,EthanS;Johnson,SheilaE;Balskus,EmilyP
肠道细菌基因毒素大肠杆菌素与结直肠癌的发展有关。在大肠杆菌素生物合成的最后阶段，无活性的前体（前大肠杆菌素）通过ClbP（一种不寻常的内膜结合周质肽酶）进行蛋白水解切割，以产生活性基因毒素。这种酶提供了监测和调节大肠杆菌素生物合成的机会，但其活性形式尚未在体外研究，并且存在有限的工具来测量其活性。在这里，我们描述了具有催化活性的全长ClbP的体外生物化学表征。我们阐明其底物的偏好，并使用这些信息来开发荧光活性探针。该工具将能够发现ClbP抑制剂并简化产大肠杆菌素细菌的鉴定。
The gut bacterial genotoxin colibactin is linked to the development of colorectal cancer. In the final stages of colibactin’s biosynthesis, an inactive precursor (precolibactin) undergoes proteolytic cleavage by ClbP, an unusual inner-membrane-bound periplasmic peptidase, to generate the active genotoxin. This enzyme presents an opportunity to monitor and modulate colibactin biosynthesis, but its active form has not been studiedin vitroand limited tools exist to measure its activity. Here, we describe thein vitrobiochemical characterization of catalytically active, full-length ClbP. We elucidate its substrate preferences and use this information to develop a fluorogenic activity probe. This tool will enable the discovery of ClbP inhibitors and streamline identification of colibactin-producing bacteria.