Predicting the emergence of antibiotic resistance by directed evolution and structural analysis
Predicting the emergence of antibiotic resistance by directed evolution and structural analysis
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DOI：
10.1038/84981
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发表时间：
2001-03-01
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影响因子：
--
通讯作者：
Stevens, RC
中科院分区：
文献类型：
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作者：
Orencia, MC;Yoon, JS;Stevens, RC
定向进化可以成为预测抗生素耐药性的有力工具。抗性涉及有益于病原体的突变的积累，同时维持对保持功能至关重要的残基相互作用和核心包装。保持稳定性的约束，同时增加活性，大大减少了可能的突变组合途径的数量。为了验证这一理论，使用超变子E.大肠杆菌为基础的定向进化技术与头孢噻肟选择。选择的突变体进行了比较，两个以前的定向进化研究和临床分离株的数据库。在所有情况下，进化导致产生E104 K/M182 T/G238 S突变组合（类似于耐药性增加500倍），相当于临床分离株TEM-52。TEM-52的结构被确定为2.4埃。G238 S将活性位点的通路扩大了2.8埃，而E104 K稳定了重组的拓扑结构。M182 T突变位于活性位点17埃处，似乎是一个全局抑制突变，其作用是稳定新的酶结构。我们的研究结果表明，定向进化结合结构分析可以用于预测未来导致抗生素耐药性增加的突变。
Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-I p-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations (similar to 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 Angstrom. G238S widens access to the active site by 2.8 Angstrom whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 Angstrom from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis fan be used to predict future mutations that lead to increased antibiotic resistance.