Directed Evolution of Mycobacterium tuberculosis β-Lactamase Reveals Gatekeeper Residue That Regulates Antibiotic Resistance and Catalytic Efficiency
Directed Evolution of Mycobacterium tuberculosis β-Lactamase Reveals Gatekeeper Residue That Regulates Antibiotic Resistance and Catalytic Efficiency
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DOI：
10.1371/journal.pone.0073123
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发表时间：
2013-09-04
期刊：
影响因子：
3.7
通讯作者：
DeLisa, Matthew P.
中科院分区：
文献类型：
--
作者：
Feiler, Christian;Fisher, Adam C.;DeLisa, Matthew P.
定向进化可以成为揭示导致更耐药细菌菌株的突变途径的有力工具。在这项研究中，我们专注于结核分枝杆菌，它对β-内酰胺类抗生素具有耐药性，因此继续构成重大的公共卫生威胁。该微生物的耐药性是组成性产生的染色体编码的超广谱A类β-内酰胺酶BlaC的结果。在此，选择氨苄青霉素上的组合酶文库以鉴定增加细菌对β-内酰胺类的抗性的突变。经过单轮诱变和选择，进化出了BlaC突变体，与野生型酶相比，其对细胞的抗生素抗性提高了5倍，并将BlaC的催化效率提高了3倍。所有分离的突变体在105位携带突变（例如，虚线垂直条105 F），其看起来将对活性位点的访问加宽了3.6埃，同时还稳定了重组的拓扑。鉴于这些发现，我们提出，断裂的垂直条105是活性位点的“看门人”残基，其调节BlaC的底物水解。此外，我们的研究结果表明，定向进化可以提供深入了解高度耐药微生物的发展。
Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium Mycobacterium tuberculosis, which is resistant to members of the beta-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A beta-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to beta-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., broken vertical bar 105F) that appears to widen access to the active site by 3.6 angstrom while also stabilizing the reorganized topology. In light of these findings, we propose that broken vertical bar 105 is a 'gatekeeper' residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.