Department of Chemistry, University of Florida , Gainesville, Florida 32611, United States.
Department of Microbial Pathogenesis, Yale School of Medicine , New Haven, Connecticut 06536, United States.
J Am Chem Soc. 2017 Dec 13;139(49):17719-17722. doi: 10.1021/jacs.7b09971. Epub 2017 Nov 28.
Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.
某些共生大肠杆菌含有 clb 生物合成基因簇，该基因簇编码称为前肠菌素的小分子前体药物。前肠菌素通过 N-去酰化转化为肠菌素；后者被推测具有遗传毒性，并有助于结直肠癌的形成。尽管在阐明（前）肠菌素生物合成方面已经取得了进展，但 clb 基因产物的几个功能和机制仍知之甚少。在这里，我们报告了 ClbS 的 2.1 Å X 射线结构和分子功能，ClbS 是一种赋予宿主细菌对肠菌素毒性抗性的基因产物，并且已被证明对细菌活力很重要。该结构包含一个潜在的肠菌素结合位点，与已知的水解酶具有相似性。使用合成的肠菌素类似物和 ClbS 或活性位点残基突变体进行的体外研究揭示了环丙烷水解酶活性，该活性将肠菌素的亲电环丙烷转化为无害的水解产物。由于环丙烷已被证明在体外对遗传毒性效应至关重要，因此这种 ClbS 催化的开环为细菌提供了一种规避自身遗传毒性的方法。我们的研究提供了第一个报道的环丙烷水解酶的分子水平视图，并支持该酶在肠菌素抗性中具有特定的机制作用。
