Orencia M C, Yoon J S, Ness J E, Stemmer W P, Stevens R C
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nat Struct Biol. 2001 Mar;8(3):238-42. doi: 10.1038/84981.
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-1 beta-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 ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A 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 can be used to predict future mutations that lead to increased antibiotic resistance.
定向进化可以成为预测抗生素耐药性的有力工具。耐药性涉及有利于病原体的突变积累，同时维持对保持功能至关重要的残基相互作用和核心堆积。在增加活性的同时保持稳定性的限制，极大地减少了可能的突变组合途径的数量。为了验证这一理论，使用基于超突变大肠杆菌的定向进化技术并结合头孢噻肟选择，对TEM-1β-内酰胺酶进行进化。将所选突变体与之前的两项定向进化研究以及临床分离株数据库进行比较。在所有情况下，进化都导致产生了E104K/M182T/G238S突变组合（耐药性增加约500倍），这与临床分离株TEM-52相当。TEM-52的结构被确定为2.4埃。G238S使进入活性位点的通道拓宽了2.8埃，而E104K稳定了重新组织的拓扑结构。M182T突变位于距活性位点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。我们的结果表明，定向进化与结构分析相结合可用于预测导致抗生素耐药性增加的未来突变。
